US20050221386A1 - Chromatographic exclusion agglutination assay and methods of use thereof - Google Patents
Chromatographic exclusion agglutination assay and methods of use thereof Download PDFInfo
- Publication number
- US20050221386A1 US20050221386A1 US11/060,113 US6011305A US2005221386A1 US 20050221386 A1 US20050221386 A1 US 20050221386A1 US 6011305 A US6011305 A US 6011305A US 2005221386 A1 US2005221386 A1 US 2005221386A1
- Authority
- US
- United States
- Prior art keywords
- porous member
- specific binding
- detectable
- binding reagents
- detectable specific
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 82
- 238000007818 agglutination assay Methods 0.000 title abstract description 23
- 230000007717 exclusion Effects 0.000 title abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 314
- 230000009870 specific binding Effects 0.000 claims abstract description 309
- 238000012360 testing method Methods 0.000 claims abstract description 185
- 238000001514 detection method Methods 0.000 claims abstract description 53
- 230000027455 binding Effects 0.000 claims abstract description 33
- 239000012491 analyte Substances 0.000 claims description 72
- 239000012530 fluid Substances 0.000 claims description 63
- 239000000203 mixture Substances 0.000 claims description 63
- 238000004891 communication Methods 0.000 claims description 54
- 239000002250 absorbent Substances 0.000 claims description 37
- 230000002745 absorbent Effects 0.000 claims description 37
- 230000009471 action Effects 0.000 claims description 31
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 22
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 22
- 239000000427 antigen Substances 0.000 claims description 19
- 108091007433 antigens Proteins 0.000 claims description 19
- 102000036639 antigens Human genes 0.000 claims description 19
- 238000002156 mixing Methods 0.000 claims description 12
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 11
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 11
- 230000002285 radioactive effect Effects 0.000 claims description 11
- 238000009825 accumulation Methods 0.000 claims description 10
- 239000007850 fluorescent dye Substances 0.000 claims description 10
- 230000015572 biosynthetic process Effects 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 230000001483 mobilizing effect Effects 0.000 claims 1
- 238000003556 assay Methods 0.000 abstract description 11
- 239000012501 chromatography medium Substances 0.000 abstract description 10
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 238000011282 treatment Methods 0.000 abstract description 4
- 238000012544 monitoring process Methods 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 240
- 239000007788 liquid Substances 0.000 description 41
- 239000000463 material Substances 0.000 description 38
- 230000004520 agglutination Effects 0.000 description 25
- 239000000020 Nitrocellulose Substances 0.000 description 23
- 229920001220 nitrocellulos Polymers 0.000 description 23
- 239000004816 latex Substances 0.000 description 21
- 229920000126 latex Polymers 0.000 description 21
- 239000012528 membrane Substances 0.000 description 21
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 16
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 16
- 150000001875 compounds Chemical class 0.000 description 16
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 15
- 239000000872 buffer Substances 0.000 description 14
- 239000002245 particle Substances 0.000 description 14
- 239000011347 resin Substances 0.000 description 13
- 229920005989 resin Polymers 0.000 description 13
- 238000011534 incubation Methods 0.000 description 12
- 239000002184 metal Substances 0.000 description 11
- 229910052751 metal Inorganic materials 0.000 description 11
- 230000000717 retained effect Effects 0.000 description 11
- 229920003023 plastic Polymers 0.000 description 10
- 239000004033 plastic Substances 0.000 description 10
- 229920002401 polyacrylamide Polymers 0.000 description 10
- 239000011148 porous material Substances 0.000 description 10
- 239000000654 additive Substances 0.000 description 8
- 229920002678 cellulose Polymers 0.000 description 8
- 239000001913 cellulose Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 229920004142 LEXAN™ Polymers 0.000 description 7
- 239000004418 Lexan Substances 0.000 description 7
- -1 antibodies Proteins 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 238000009736 wetting Methods 0.000 description 7
- 206010041925 Staphylococcal infections Diseases 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 201000004792 malaria Diseases 0.000 description 6
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 210000002700 urine Anatomy 0.000 description 6
- 229920003043 Cellulose fiber Polymers 0.000 description 5
- 229920000297 Rayon Polymers 0.000 description 5
- 239000000919 ceramic Substances 0.000 description 5
- 239000002131 composite material Substances 0.000 description 5
- 229920001577 copolymer Polymers 0.000 description 5
- 150000001925 cycloalkenes Chemical class 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 239000003365 glass fiber Substances 0.000 description 5
- 230000005484 gravity Effects 0.000 description 5
- 150000002739 metals Chemical class 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 239000000123 paper Substances 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 239000010453 quartz Substances 0.000 description 5
- 239000002964 rayon Substances 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 229910052710 silicon Inorganic materials 0.000 description 5
- 239000010703 silicon Substances 0.000 description 5
- 239000000377 silicon dioxide Substances 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 239000012190 activator Substances 0.000 description 4
- 239000003638 chemical reducing agent Substances 0.000 description 4
- 238000005345 coagulation Methods 0.000 description 4
- 230000015271 coagulation Effects 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 239000003599 detergent Substances 0.000 description 4
- 229920001971 elastomer Polymers 0.000 description 4
- 239000002532 enzyme inhibitor Substances 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 239000007800 oxidant agent Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000005060 rubber Substances 0.000 description 4
- 239000003381 stabilizer Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 150000008163 sugars Chemical class 0.000 description 4
- 210000000601 blood cell Anatomy 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229920000728 polyester Polymers 0.000 description 3
- 238000005086 pumping Methods 0.000 description 3
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 241000193985 Streptococcus agalactiae Species 0.000 description 2
- 101000588258 Taenia solium Paramyosin Proteins 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000011533 pre-incubation Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 108091023037 Aptamer Proteins 0.000 description 1
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241001591005 Siga Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000000991 chicken egg Anatomy 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- UVCJGUGAGLDPAA-UHFFFAOYSA-N ensulizole Chemical compound N1C2=CC(S(=O)(=O)O)=CC=C2N=C1C1=CC=CC=C1 UVCJGUGAGLDPAA-UHFFFAOYSA-N 0.000 description 1
- 239000000576 food coloring agent Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229960003085 meticillin Drugs 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- 229920009537 polybutylene succinate adipate Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
- G01N33/5304—Reaction vessels, e.g. agglutination plates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
- G01N33/54389—Immunochromatographic test strips based on lateral flow with bidirectional or multidirectional lateral flow, e.g. wherein the sample flows from a single, common sample application point into multiple strips, lanes or zones
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7769—Measurement method of reaction-produced change in sensor
- G01N2021/7786—Fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Definitions
- the present invention relates generally to the field of agglutination assays, and particularly to chromatographic exclusion assays and methods of use thereof.
- a variety of air agglutination assays are commercially available. These agglutination assays can be used for a variety of purposes, such as diagnosing conditions of disease or monitor treatments by analyzing trace analytes of interest in a sample. Certain agglutination assays are fully performed in solution, with the presence of visible analyte induced aggregates in the solution as indication of positive result. Other agglutination assays are performed utilizing a flow-through system where a reaction sample is passed through a small hole in order to concentrate analyte induced aggregates. Other agglutination assays are performed on dry porous strips, with the presence of anlalyte induced aggregates on the strip as indication of positive result.
- agglutination assays suffer from lack of sensitivity and are prone to providing false-positive results.
- the agglutination assays that utilize a dry porous strip are especially prone to providing false-positive results because of difficulties in observing and detecting true agglutinates that are aggregated in or on the porous strip from random clumping of the agglutination assay components.
- Most agglutination assays, especially the flow-through agglutination assays are prone to providing false-positive results because of the presence of non-specific particulate matter in a sample under test.
- agglutination assays usually require a highly skilled person to determine the end result accurately as positive or negative, or to make the decision to run the test again for a more definitive result. While there has been a trend to produce more sensitive agglutination assays, further improvements in the sensitivity, effectiveness, and simplicity for home or clinic use of agglutination assays are desirable, and the present invention addresses the existing problems and provides related benefits.
- the present invention relates to agglutination assays and in particular to chromatographic exclusion assays and methods of use thereof.
- the present invention includes a novel strategy for determining the presence of one or more analytes on interest in a test sample by using chromatographic exclusion or separation of aggregates of bound detectable specific binding reagents that are accumulated at a particular and non-random location on the test device.
- the specific detectable binding reagent aggregates are not formed, and hence not excluded from the chromatographic media creating a distinct and readily differentiating event.
- the present invention is particularly adaptable as a simple test device for detection of diseases or monitoring of treatments at a doctor's office or in the home.
- agglutination assays can be made more sensitive, more efficient, and more simple using chromatographic exclusion or separation of aggregates formed by the binding of a detectable specific binding reagent with the analytes of interest.
- the chromatographically excluded or separated aggregates of a positive test result lead to a distinct event, such as the formation of a visible band on the chromatographic media, which is easily differentiated from the result of a negative test where no aggregates are excluded or separated from the chromatographic media.
- a first aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a test sample including a first porous member that includes one or more specific binding reagents that are capable of being detected and capable of binding to the analyte or analytes of interest suspected of being present in a test sample.
- the present invention also includes a second porous member that is in fluid communication with the first porous member, and is capable of substantially allowing any unbound detectable specific binding reagent to pass through freely, but substantially retaining any bound detectable specific binding reagent.
- the sample moves through the first and second porous member by capillary flow and the presence of the analyte or analytes of interest in the test sample is indicated by the presence of bound detectable specific binding reagent aggregates at the interface of the first porous member with the second porous member.
- a second aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a sample including a first porous member and a second porous member in fluid communication with one another forming a detection zone at the junction of the first and second porous members.
- a reaction zone positioned on the first porous member includes one or more detectable specific binding reagents that are able to bind to the analyte or analytes of interest suspected of being present in the sample under test and form aggregates.
- the one or more detectable specific binding reagents present at the reaction zone are capable of solublizing and moving to the second porous member by capillary flow.
- the second porous member is capable of substantially allowing any unbound detectable specific binding reagent to pass through freely, but substantially impeding the flow of any bound and aggregated detectable specific binding reagent.
- the sample moves through the first and second porous member by capillary flow and the presence of the analyte or analytes of interest in the test sample is indicated by the formation of an accumulation of bound detectable specific binding reagent aggregates at the junction of the first porous member with the second porous member, and the absence of the analyte or analytes of interest in the test sample is indicated by the absence or lack of an accumulation of bound detectable specific binding reagent aggregates at the junction of the first porous member with the second porous member.
- a third aspect of the present invention includes a method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of: providing a first porous member that includes one or more detectable specific binding reagents that are able to bind to the analyte or analytes of interest suspected of being present in the test sample; providing a second porous member, which is in fluid communication with the first porous member and forming a junction, such that the second porous member substantially allows any unbound detectable binding reagent to pass through freely, but substantially prevents the flow of any bound detectable specific binding reagent; introducing the test sample to the first porous member, such that the test sample moves through the first and second porous members by capillary flow and mobilizes the one or more detectable specific binding reagents of the first porous member; wherein the presence of the analyte or analytes of interest is indicated by the presence of bound detectable specific binding reagent aggregates at the junction of the first porous member with the second por
- a fourth aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a sample including one or more detectable specific binding reagents capable of being detected and capable of binding to the one or more analytes of interest when added to the sample.
- the present invention also includes a first porous member and a second porous member in fluid communication with one another wherein the second porous member is capable of substantially allowing the one or more detectable specific binding reagents to pass through freely, but substantially retaining the detectable specific binding reagents when they are bound to an analyte or analytes of interest.
- the one or more detectable specific binding reagents are introduced to the sample forming a mixture and the mixture is then introduced to the first porous member, moving through the first and second porous members via capillary action, and the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagent aggregates at the interface of the first porous member with the second porous member.
- a fifth aspect of the present invention includes a method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of: mixing one or more detectable specific binding reagents able to bind to said one or more analytes of interest in the sample with the sample in order to form a mixture; providing a first porous member; providing a second porous member in fluid communication with the first porous member and forming a junction.
- the second porous member being capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the second porous member, but substantially preventing the flow of the one or more detectable specific binding reagents through the second porous member when bound to the analyte or analytes of interest; introducing the mixture to the first porous member, wherein the mixture moves by capillary action through the first and the second porous members; wherein, the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagents aggregates at the junction of the first and second porous members.
- a sixth aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a sample including a first porous member including one or more detectable specific binding reagents capable of being detected and capable of binding to the one or more analytes of interest when added to the sample.
- the present invention also includes a second and a third porous members wherein the third porous member is in fluid communication with the second porous member, and the third porous member is capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the third porous member, but substantially retaining the one or more detectable specific binding reagents when the one or more detectable specific binding reagents are bound to the one or more analytes of interest; whereby, the sample is introduced to the first porous member and the first porous member is brought into fluid contact with the second porous member, wherein the sample moves through the first, second, and third porous members by capillary flow and, wherein the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagents aggregates at the interface of the second porous member with the third porous member.
- a seventh aspect of the present invention includes a method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of: providing a first porous member; providing a second porous member; providing a third porous member in fluid communication with the second porous member forming a junction, and the third porous member being capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the third porous member, but substantially preventing the flow of the one or more detectable specific binding reagents through the third porous member when bound to the one or more analytes of interest; mixing one or more detectable specific binding reagents able to bind to the one or more analytes of interest in the sample with the sample forming a mixture; introducing the mixture to the first porous member; contacting the first porous member with the second porous member wherein the mixture moves by capillary action through the first, second, and third porous members; wherein, the presence of the one or more analytes of interest is indicated by the
- An eighth aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a test sample including one or more detectable specific binding reagents that are capable of being detected and capable of binding to the analyte or analytes of interest suspected of being present in a test sample.
- the present invention also includes a porous member having a first end and a second end, and is capable of substantially allowing any unbound detectable specific binding reagent to pass through freely, but substantially excluding any bound detectable specific binding reagent.
- the sample When the sample is mixed with the one or more detectable binding agents and is introduced to the first end of the porous member of the present invention, the sample moves through the porous member by capillary flow and the presence of the analyte or analytes of interest in the test sample is indicated by the substantial absence of bound detectable specific binding reagent aggregates at the second end of the porous member which were excluded from the porous member at first end of the porous member.
- a ninth aspect of the present invention includes method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of: mixing one or more detectable specific binding reagents able to bind to said one or more analytes of interest in the sample with the sample in order to form a mixture; providing a porous member having a first end and a second end and capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the porous member, but substantially preventing the flow of the one or more detectable specific binding reagents through the porous member when they are bound to the analyte or analytes of interest; introducing the mixture to the first end of the porous member, wherein the mixture moves by capillary action through the porous member; wherein, the presence of the one or more analytes of interest is indicated by the substantial absence of the bound one or more detectable specific binding reagents aggregates at the second end of the porous member that were excluded from the porous member at the first end of the
- FIG. 1A is a top view of an embodiment of the present invention.
- FIG. 2B is a top view of an embodiment of the present invention.
- agglutination assays can be made more sensitive, more efficient, and more simple using chromatographic exclusion or separation of aggregates formed by the binding of one or more detectable specific binding reagents with the analyte or analytes of interest at a particularly designated and non-random location on the test device.
- the chromatographically excluded aggregates of a positive test result lead to a distinct event, such as the formation of a visible band on the chromatographic media, which is easily differentiated from the result of a negative test where no aggregates are excluded form the chromatographic media.
- the present invention includes several general and useful aspects, including:
- the present invention relates to agglutination assays and in particular to chromatographic exclusion assays and methods of use thereof
- the present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion or separation of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device.
- the particular arrangement of the chromatographic media of the present invention is utilized in order to increase sensitivity and eliminate subjective interpretation of the test result.
- the present invention provides an exclusion of aggregate that is definitively detectable when the analyte or analytes of interest are present in the sample, and in the absence of the analyte or analytes of interest in the sample under test, the detectable specific binding reagent aggregates are not formed at the detection zone of the test device, and hence, are not excluded from the chromatographic media, which creates a distinct and readily differentiating event.
- the present invention is particularly adaptable as a simple and rapid test device for detection of diseases or monitoring of treatments at a doctor's office or in the home.
- the present invention includes a first porous member, which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- a first porous member which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- Such material include, but are not limited to, glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- the first porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the first porous member, whether the detectable specific binding reagents are bound to analytes of interest or not Furthermore the pores or channels of the first porous member should be large enough as to not substantially impede or retard the free flow of any coagulated or aggregates of bound detectable specific binding reagent through the first porous member.
- the first porous member may have pores or channels of any size, which will be largely dependent on the size of the specific binding reagent being used or any particle or label which may be used as a label.
- the pore size of the first porous member may be, for example, about 10 micrometers to about 500 micrometer, and preferably about 50 micrometers to about 200 micrometers.
- the present invention also includes a second porous member that is in fluid communication with the first porous member of the present invention.
- a second porous member that is in fluid communication with the first porous member of the present invention.
- one end of the first porous member is in contact and joins one end of the second porous member, forming a junction where the first porous member ends and the second porous member begins in fluid contact.
- Any aqueous or liquid sample that contacts the first porous member will be moved by capillary action towards the second porous member where it moves past the junction of the first and second porous members and continues to moves through the second porous member.
- the second porous member of the present invention can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- Such material include, but are not limited to, glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- An important aspect of the second porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the second porous member only when the detectable specific binding reagent is not bound to any analyte. If the one or more analytes of interest are present in the test sample, binding events occur between the one or more detectable specific binding reagents of the present invention and the one or more analytes of interest of the test sample and complexes of detectable specific binding reagents and analytes are formed.
- the second porous member substantially arrests or impedes the flow of any bound detectable specific binding reagent, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of detectable specific binding reagents complexes.
- the second porous member may have pores or channels of any size, which will be largely dependent on the size of the specific binding reagent being used or any particle or label which may be used as a label. Because the second porous member must substantially allow unbound detectable specific binding reagents to flow freely through the second porous member, the pore size of the second porous member may be, for example, about 0.1 micrometers to about 100 micrometers, and preferably about 1 micrometer to about 20 micrometers.
- the capillary action provides the driving source or pumping force of the movement of the liquid through the device.
- the device is normally utilized in the horizontal position so that the capillary flow of the liquid sample through the device is normally a lateral flow, which is not affected by the force of gravity.
- certain embodiments of the present invention may include added driving forces, such as for example, gravity or certrifugal force.
- the detectable specific binding reagents of the present invention are preferably substantially freely mobile when in the solublized or moist state.
- each type of detectable specific binding regent of the first porous member is capable of binding a single analyte of interest suspected of being present in the test sample forming aggregates, and each type of detectable specific binding reagent is distinguishable from one another.
- the detectable specific binding reagents can be any anti-analyte, namely a compound that is capable of specifically binding to an analyte of interest forming aggregates.
- the analytes of interest may include, for example, any material capable of being detected by a specific binding reagent including any cells or viruses or any components thereof.
- the analytes of interest may also include any small organic molecules, such as drugs, hormones, steroids, neurotransmitters, growth factors, metabolites, or other chemicals.
- the analytes of interest may include, for example, any large organic molecules, such as nucleic acids, proteins, polysaccharides, or other large molecules.
- the detectable specific binding reagent of the present invention may be, for example, an intact antibody, such as a polyclonal or a monoclonal antibody.
- the detectable specific binding regent is an antibody, for example, human or other mammalian IgG, IgG 1 IgG 2 , IgG 3 , IgG 4 , IgM, IgA, IgY, SigA, or IgE.
- the detectable specific binding reagent of the present invention may also be an antibody fragment, such as an antigen-binding fragment or Fab, or an F(ab′) 2 fragment, or the antigen-binding site of an antibody (such as a complementarity-determining region of an antibody).
- the detectable specific binding reagent is preferably a purified, high affinity monoclonal antibody that specifically binds an analyte of interest suspected of being present in the test sample.
- the detectable specific binding reagent may, in other embodiments, be an antigen, a ligand, or a receptor.
- the detectable specific binding reagent may bind to more than one type of analyte, but preferably binds only one type of analyte that is suspected of being present in the test sample.
- the detectable specific binding reagent may be comprised of more than one reagent; for example, the detectable specific binding reagent may comprise more than one type of monoclonal antibody or antibody fragment, each of which specifically binds the same type of analyte of interest suspected of being present in the test sample.
- the detectable specific binding reagent of the present invention can include, without limitation, peptides, polypeptides, antibodies, Fab fragments, fusion proteins, chimeric or hybrid molecules, nucleic acids, nucleic acid mimics (for example, peptide nucleic acids), carbohydrates, cell surface antigens, receptors, ligands, or combinations thereof
- the detectable specific binding reagent includes an antibody (monoclonal or polyclonal, natural, modified, or recombinant) or an antibody fragment (such as an Fab fragment or single-chain antibody variable region fragment).
- the detectable specific binding reagent can include an antigen, such as an antigen capable of specifically binding to an antibody that recognizes an analyte of interest.
- the detectable binding reagent can include a nucleic acid or nucleic acid mimic aptamer that binds a target such as a peptide or small molecule, or a receptor that binds a ligand, or a ligand that binds a receptor.
- the detectable specific binding reagent may be capable of binding to a mimotope, such as a peptide, that mimics an analyte of interest (see, for example, Kieber-Emmons (1998) Immunol. Res., 17:95-108; Shin et al. (2001) Infect.
- the detectable specific binding reagent also includes the capacity to be detected and preferably each type of detectable specific binding reagent is distinguishably detectable from one another.
- the detectable specific binding reagents' capacity to be detected may be any quality capable of being detected, and includes, for example, detection by a label such as a color label including dyes, metal sols, and latex particles, or capacity to be detected by a label such as, for example, fluorescent label, radioactive label, magnetic label or chemiluminescent label.
- the detectable specific binding reagents' capacity to be detected may also be, for example, detection by size, charge, polarity, hydrophobicity, hydrophilicity, lipophilicity, or viscosity.
- each detectable specific binding reagent that is specific for a certain analyte of interest may include a different capacity to be detected, for example, different color labels, different fluorescent labels, or any differentiating capacity to be distinguished, or any combination thereof.
- Detection methods have enormous diversity and are presently available, and any detection method may be employed depending on the protocol.
- the detection methods may include, for example, direct detection of labels such as metal sols, colored labels, colored beads, colored particles, or fluorescent labels, either by way of direct vision or by way of a microscope.
- Labels of the present invention may be of any size and it will be largely dependent on the test that is to be performed and the pore size or chanel size of the porous members of the present invention.
- the labels of the present invention may have a size range of, for example, about 0.01 micrometers in diameter to about 50 micrometers in diameter, and preferably about 0.02 micrometers in diameter to about 2 micrometers in diameter.
- the detection methods may also include, for example, indirect detection of detectable labels such as magnetic labels, radioactive labels, or the measurement of scattered light to measure a change in the size population.
- the present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device.
- the present invention includes a first porous member including one or more detectable specific binding reagents that are each capable of being detected and each capable of biding to an analyte of interest in the test sample.
- the detectable specific binding reagents of the first porous members are preferably dried onto or otherwise temporarily immobilized into a reaction zone or region of the first porous member such that the reaction zone of the first porous member is impregnated by the dried detectable specific binding reagents.
- the impregnated detectable specific binding reagents of the first porous member are preferably substantially freely mobile when in the moist or solublized state.
- first porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the first porous member, whether the detectable specific binding reagents are bound to analytes of interest or not. Furthermore the pores of the first porous member should be large enough as to not substantially impede the free flow of any coagulated or aggregates of bound detectable specific binding reagent through the first porous member.
- the present invention includes a second porous member that is in fluid communication with the first porous member of the present invention.
- one end of the first porous member is in contact and joins one end of the second porous member, forming a junction where the first porous member ends and the second porous member begins in fluid contact.
- Any aqueous or liquid sample that contacts the first porous member will be moved by capillary action towards the second porous member where it moves past the junction of the first and second porous members and continues to moves through the second porous member.
- An important aspect of the second porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the second porous member only when the detectable specific binding reagent is not bound to any analyte.
- the second porous member substantially retains and impedes the flow of any bound detectable specific binding reagent, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents.
- the presence or absence of one or more analytes in a test sample is determined by contacting the test sample to the first porous member of the present invention.
- the test sample moves through the first porous member by capillary flow where the one or more detectable specific binding reagents of the first porous member are solubilized and are mixed in with the test sample forming a mixture. If one or more analytes of interest are present in the test sample, the one or more detectable specific binding reagents bind the one or more analytes of interest forming a complex, and the detectable specific binding reagents analyte complexes move freely through the first porous member by capillary flow towards the second porous member of the present invention.
- the bound detectable specific binding reagents analyte complexes are substantially retained by the second porous member, which does not substantially allow bound detectable specific binding reagents analyte complexes to pass freely through the second porous member. Determining the presence of aggregated bound detectable specific binding reagents analyte complexes that are substantially accumulated at the junction of the first and second porous members indicates the presence of one or more analytes of interest in the sample.
- the one or more detectable specific binding reagents are distinguished based on, for example, the color of different labels or the fluorescence of different labels designated for the detection and correlating to a specific analyte of interest, then the identity of each of the one or more analytes of interest suspected of being present in the test sample are revealed.
- the one or more analytes of interest are not present in the test sample, substantially no detectable specific binding reagents analyte complexes are formed and hence are not retained by the second porous member, which allows any unbound detectable specific binding reagents to pass freely through the second porous member.
- coagulation, agglutination, precipitation, accumulation, clumping, or plug formation by any means or as appropriate, increasing viscosity can be used as part of the detection scheme.
- control zone located on the second porous member in order to ensure a test has been completed.
- the control zone is located on the second porous member at some distance away from the junction of the first and second porous members.
- the control zone may include a region on the second porous member at some distance away from the junction of the first and second porous members, where it can indicate that the test sample has travelled past the detection zone or the junction of the first and second porous members.
- a control compound can be used, for example a colored solution, which can be mixed in with the test sample and be detected at the control zone indicating that the test sample has travelled through the device past the junction of the first and second porous members.
- the control compound may also be, for example a control detectable label such as control latex.
- the control zone can include immobilized specific biding reagents that are capable of detecting a control compound.
- the control compound may include for example, a known substance that does not bind to the detectable specific binding regents of the first porous member.
- the control compound can be mixed in with the sample, which will pass through the first and second porous members without being retained at the junction of the first and second porous member and reach the control zone where it will bind to the immobilized specific binding reagent of the control zone, indicating that the test sample has travelled through the test device and has passed the junction of the first and second porous members.
- additives may be included in the present invention for specific purposes, such as for example, buffers, enzyme inhibitors, enzyme substrate or cofactors, preservatives, stabilizers, solublizing agents, detergents, sugars, facilitators, activators, oxidants, reductants, or any other additives desired for a particular purpose.
- an absorbent pad in fluid communication with the first porous member of the present invention.
- the absorbent pad can be made of any material capable of holding an aqueous or liquid sample in fluid communication with the first porous member such that the sample can flow evenly through the first porous member by capillary action.
- the present invention may also include an absorbent pad in fluid communication with the distal end of the second porous member, having sufficient capacity to absorb any liquid and acting as a distal sink at the end of the test device in order to remove any excess liquid sample from the components of the present invention.
- the present invention may also include, for example, a filter positioned between the first porous member and the second porous member and in a continuous fluid communication with the first and second porous members.
- the filter may act to filter out or trap larger components above a certain desired range, which may be present in the test sample. For example, if the test sample includes a blood sample, the filter may be used to filter out blood cells or other components of blood larger than a desired size.
- the present invention is adaptable to be used with a plurality of similar first porous member and second porous member in fluid communication as described above. These plurality of porous members may be used, for example to run different desired tests on the same test sample at the same time. These plurality of porous members may be used in parallel configuration or stacked on top of one another.
- the housing may be made of any rigid material capable of housing the components of the present invention
- the housing may be preferably made of an impermeable material that contains the liquid sample and the components of the present invention inside the device without allowing for any leaks for permeation.
- the housing may be made of, for example, plastic, glass, metal, rubber, or any other non-permeable material.
- the housing may preferably include one or more apertures.
- the housing may include, for example, an aperture located on the area of the first porous member to act as an inlet for introduction of the test sample to the first porous member, located at some distance away from the junction of the first and second porous member.
- the housing may also include an aperture located on the area of the detection zone or the junction of the first and second porous members to allow for detection of whether the test result turned positive or negative.
- the housing may also include an aperture located on the area of the control zone, located on the second porous member at some distance away from the detection zone or the junction of the first and second porous members to allow for detection of whether or not the test has reached completion.
- the present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device.
- the present invention includes one or more detectable specific binding reagents that are each capable of being detected and each capable of binding to an analyte of interest in the test sample when added to the test sample.
- the detectable specific binding reagents of the present invention may be separately mixed with the test sample suspected of containing one or more analytes of interest forming a mixture, and incubated for a time period sufficient to allow the one or more detectable specific binding reagents to bind the one or more analytes of interest in the test sample. After proper incubation, the mixture of test sample and detectable specific binding reagents is used to run through the test device of the present invention.
- the present invention includes a first porous member, which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- first porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the first porous member, whether the detectable specific binding reagents are bound to analytes of interest or not. Furthermore the pores of the first porous member should be large enough as to not substantially impede the free flow of any coagulated or aggregates of bound detectable specific binding reagent through the first porous member.
- the present invention includes a second porous member that is in fluid communication with the first porous member of the present invention.
- one end of the first porous member is in contact and joins one end of the second porous member, forming a junction where the first porous member ends and the second porous member begins in fluid contact.
- Any aqueous or liquid sample that contacts the first porous member will be moved by capillary action towards the second porous member where it moves pass the junction of the first and second porous members and continues to moves through the second porous member.
- An important aspect of the second porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the second porous member only when the detectable specific binding reagent is not bound to any analyte.
- the second porous member substantially retains and impedes the flow of any bound detectable specific binding reagent, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents.
- a test sample may be tested by separately mixing the test sample suspected of containing one or more analytes of interest with the detectable specific binding reagents of the present invention forming a mixture, and incubated for a time period sufficient to allow the one or more detectable specific binding reagents to bind the one or more analytes of interest in the test sample.
- the presence or absence of one or more analytes in a test sample is determined by contacting the test sample and detectable specific binding reagent mixture to the first porous member of the present invention.
- the one or more detectable specific binding reagents bind the one or more analytes of interest forming a complex in the test mixture, and the detectable specific binding reagents analyte complexes move freely through the first porous member by capillary flow towards the second porous member of the present invention.
- the bound detectable specific binding reagents analyte complexes are substantially retained by the second porous member, which does not substantially allow bound detectable specific binding reagents analyte complexes to pass freely through the second porous member.
- Determining the presence of aggregated bound detectable specific binding reagents analyte complexes that are substantially accumulated at the junction of the first and second porous members indicates the presence of one or more analytes of interest in the sample. Furthermore, if the one or more detectable specific binding reagents are distinguished based on, for example, the color of different labels or the fluorescence of different labels designated for the detection and correlating to a specific analyte of interest, then the identity of each of the one or more analytes of interest suspected of being present in the test sample are revealed.
- substantially no detectable specific binding reagents analyte complexes are formed and hence are not retained by the second porous member, which allows any unbound detectable specific binding reagents to pass freely through the second porous member.
- coagulation, agglutination, precipitation, accumulation, clumping, or plug formation by any means or as appropriate, increasing viscosity can be used as part of the detection scheme.
- control zone located on the second porous member in order to ensure a test has been completed.
- the control zone is located on the second porous member at some distance away from the junction of the first and second porous members.
- the control zone may include a region on the second porous member at some distance away from the junction of the first and second porous members, where it can indicate that the test sample has travelled past the detection zone or the junction of the first and second porous members.
- a control compound can be used, for example a colored solution, which can be mixed in with the test sample and be detected at the control zone indicating that the test sample has travelled through the device past the junction of the first and second porous members.
- the control compound may also be, for example a control detectable label such as control latex.
- the control zone can include immobilized specific biding reagents that are capable of detecting a control compound.
- the control compound may include for example, a known substance that does not bind to the detectable specific binding regents used to test the test sample.
- the control compound can be mixed in with the sample, which will pass through the first and second porous members without being retained at the junction of the first and second porous member and reach the control zone where it will bind to the immobilized specific binding reagent of the control zone, indicating that the test sample has travelled through the test device and has passed the junction of the first and second porous members.
- additives may be included in the present invention for specific purposes, such as for example, buffers, enzyme inhibitors, enzyme substrate or cofactors, preservatives, stabilizers, solublizing agents, detergents, sugars, facilitators, activators, oxidants, reductants, or any other additives desired for a particular purpose.
- an absorbent pad in fluid communication with the first porous member of the present invention.
- the absorbent pad can be made of any material capable of holding an aqueous or liquid sample in fluid communication with the first porous member such that the sample can flow evenly through the first porous member by capillary action.
- the present invention may also include an absorbent pad in fluid communication with the distal end of the second porous member, having sufficient capacity to absorb any liquid and acting as a distal sink at the end of the test device in order to remove any excess liquid sample from the components of the present invention.
- the present invention may also include, for example, a filter positioned between the first porous member and the second porous member and in a continuous fluid communication with the first and second porous members.
- the filter may act to filter out or trap larger components above a certain desired range, which may be present in the test sample. For example, if the test sample includes a blood sample, the filter may be used to filter out blood cells or other components of blood larger than a desired size.
- the housing may be made of any rigid material capable of housing the components of the present invention
- the housing may be preferably made of an impermeable material that contains the liquid sample and the components of the present invention inside the device without allowing for any leaks for permeation.
- the housing may be made of, for example, plastic, glass, metal, rubber, or any other non-permeable material.
- the housing may preferably include one or more apertures.
- the housing may include, for example, an aperture located on the area of the first porous member to act as an inlet for introduction of the test sample to the first porous member, located at some distance away from the junction of the first and second porous member.
- the housing may also include an aperture located on the area of the detection zone or the junction of the first and second porous members to allow for detection of whether the test result turned positive or negative.
- the housing may also include an aperture located on the area of the control zone, located on the second porous member at some distance away from the detection zone or the junction of the first and second porous members to allow for detection of whether or not the test has reached completion.
- the present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device.
- the analytes of interest may include any analyte capable of being detected.
- the present invention includes a first porous member, which can be made of and include any type of material, that is capable of retaining liquid and allowing the liquid to be transported away by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- a first porous member which can be made of and include any type of material, that is capable of retaining liquid and allowing the liquid to be transported away by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- Such material include, but are not limited to, glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- the present invention includes a second porous member, which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- a second porous member which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- Such material include glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- An important aspect of the second porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the first porous member, whether the detectable specific binding reagents are bound to analytes of interest or not. Furthermore the pores of the first porous member should be large enough as to not substantially impede the free flow of any coagulated or aggregates of bound detectable specific binding reagent through the first porous member.
- the present invention includes a third porous member that is in fluid communication with the second porous member of the present invention.
- one end of the second porous member is in contact and joins one end of the third porous member, forming a junction where the second porous member ends and the third porous member begins in fluid contact.
- Any aqueous or liquid sample that contacts the second porous member will be moved by capillary action towards the third porous member where it moves past the junction of the second and third porous members and continues to moves through the third porous member.
- the third porous member of the present invention can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- An important aspect of the third porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the third porous member only when the detectable specific binding reagent is not bound to any analyte.
- the third porous member substantially retains and impedes the flow of any bound detectable specific binding reagent, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents.
- the presence or absence of one or more analytes in a test sample is determined by contacting the test sample to the first porous member of the present invention.
- the test sample is then retained by the first porous member where the one or more detectable specific binding reagents of the first porous member are solubilized and are mixed in with the test sample forming a mixture. If one or more analytes of interest are present in the test sample, the one or more detectable specific binding reagents bind the one or more analytes of interest forming a complex.
- detectable specific binding reagents analyte complexes move freely through from the first porous member to the second porous member by capillary flow towards the third porous member of the present invention.
- the bound detectable specific binding reagents analyte complexes are substantially retained by the third porous member, which does not substantially allow bound detectable specific binding reagents analyte complexes to pass freely through the third porous member.
- Determining the presence of aggregated bound detectable specific binding reagents analyte complexes that are substantially accumulated at the junction of the second and third porous members indicates the presence of one or more analytes of interest in the sample. Furthermore, if the one or more detectable specific binding reagents are distinguished based on, for example, the color of different labels or the fluorescence of different labels designated for the detection and correlating to a specific analyte of interest, then the identity of each of the one or more analytes of interest suspected of being present in the test sample are revealed.
- substantially no detectable specific binding reagents analyte complexes are formed and hence are not retained by the second porous member, which allows any unbound detectable specific binding reagents to pass freely through the second porous member.
- coagulation, agglutination, precipitation, accumulation, clumping, or plug formation by any means or as appropriate, increasing viscosity can be uses as part of the detection scheme.
- control zone located on the third porous member in order to ensure a test has been completed.
- the control zone is located on the third porous member at some distance away from the junction of the second and third porous members.
- the control zone may include a region on the third porous member at some distance away from the junction of the second and third porous members, where it can indicate that the test sample has travelled past the detection zone or the junction of the second and third porous members.
- a control compound can be used, for example a colored solution, which can be mixed in with the test sample and be detected at the control zone indicating that the test sample has travelled through the device past the junction of the second and third porous members.
- the control compound may also be, for example a control detectable label such as control latex.
- the control zone can include immobilized specific biding reagents that are capable of detecting a control compound.
- the control compound may include for example, a known substance that does not bind to the detectable specific binding regents of the first porous member.
- the control compound can be mixed in with the sample, which will pass from the first porous member to the second porous member upon contact and moves through the second and third porous members without being retained at the junction of the second and third porous member and reach the control zone where it will bind to the immobilized specific binding reagent of the control zone, indicating that the test sample has travelled through the test device and has passed the junction of the second and third porous members.
- additives may be included in the present invention for specific purposes, such as for example, buffers, enzyme inhibitors, enzyme substrate or cofactors, preservatives, stabilizers, detergents, sugars, facilitators, activators, oxidants, reductants, or any other additives desired for a particular purpose.
- an absorbent pad in fluid communication with the first porous member or the second porous member of the present invention.
- the absorbent pad can be made of any material capable of holding an aqueous or liquid sample in fluid communication with the first porous member or the second porous member. If the absorbent pad is positioned in fluid contact with the first porous member, the sample can be retained evenly through the first porous member and be transferred evenly to the second porous member upon contact and flow evenly through the first porous member by capillary action. If the absorbent pad is positioned in fluid contact with the second porous member, the sample can be flow evenly through the second porous member and into the second porous by capillary action.
- the present invention may also include an absorbent pad in fluid communication with the distal end of the third porous member, having sufficient capacity to absorb any liquid and acting as a distal sink at the end of the test device in order to remove any excess liquid sample from the components of the present invention.
- the present invention may also include, for example, a filter positioned between the second porous member and the third porous member and in a continuous fluid communication with the first and second porous members.
- the filter may act to filter out or trap larger components above a certain desired range, which may be present in the test sample. For example, if the test sample includes a blood sample, the filter may be used to filter out blood cells or other components of blood larger than a desired size.
- the present invention is adaptable to be used with a plurality of similar second porous member and third porous member in fluid communication as described above. These plurality of porous members may be used, for example to run different desired tests on the same test sample at the same time. These plurality of porous members may be used in parallel configuration or stacked on top of one another.
- the housing may be made of any rigid material capable of housing the components of the present invention
- the housing may be preferably made of an impermeable material that contains the liquid sample and the components of the present invention inside the device without allowing for any leaks for permeation.
- the housing may be made of, for example, plastic, glass, metal, rubber, or any other non-permeable material.
- the housing may preferably include one or more apertures.
- the housing may include, for example, an aperture located on the area of the second porous member to act as an inlet for introduction of the test sample to the second porous member, located at some distance away from the junction of the second and third porous member.
- the housing may also include an aperture located on the area of the detection zone or the junction of the second and third porous members to allow for detection of whether the test result turned positive or negative.
- the housing may also include an aperture located on the area of the control zone, located on the third porous member at some distance away from the detection zone or the junction of the second and third porous members to allow for detection of whether or not the test has reached completion.
- the present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device.
- the analytes of interest may include any analyte capable of being detected.
- the present invention includes one or more detectable specific binding reagents that are each capable of being detected and each capable of binding to an analyte of interest in the test sample when added to the test sample.
- the detectable specific binding reagents of the present invention may be separately mixed with the test sample suspected of containing one or more analytes of interest forming a mixture, and incubated for a time period sufficient to allow the one or more detectable specific binding reagents to bind the one or more analytes of interest in the test sample. After proper incubation, the mixture of test sample and detectable specific binding reagents is used to run through the, test device of the present invention.
- the present invention includes a porous member having a first end and a second end, which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- material include glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- the capillary action provides the driving source or pumping force of the movement of the liquid through the device.
- the device is normally utilized in the horizontal position so that the capillary flow of the liquid sample through the device is normally a lateral flow, which is not affected by the force of gravity.
- porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the porous member only when the detectable specific binding reagent is not bound to any analyte.
- the porous member substantially excludes any bound detectable specific binding reagent from the porous member, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents.
- the capillary action provides the driving source or pumping force of the movement of the liquid through the device.
- the device is normally utilized in the horizontal position so that the capillary flow of the liquid sample through the device is normally a lateral flow, which is not affected by the force of gravity.
- certain embodiments of the present invention may include added driving forces, such as for example, gravity or certrifugal force.
- a test sample may be tested by separately mixing the test sample suspected of containing one or more analytes of interest with the detectable specific binding reagents of the present invention forming a mixture, and incubated for a time period sufficient to allow the one or more detectable specific binding reagents to bind the one or more analytes of interest in the test sample.
- the presence or absence of one or more analytes in a test sample is determined by contacting the test sample and detectable specific binding reagent mixture to the first end of the porous member of the present invention.
- the one or more detectable specific binding reagents bind the one or more analytes of interest forming a complex in the test mixture, and the detectable specific binding reagents analyte complexes are substantially excluded from the porous member, which does not substantially allow bound detectable specific binding reagents analyte complexes to pass freely through the second porous member. Determining the presence of aggregated bound detectable specific binding reagents analyte complexes that are substantially accumulated at the first end of the porous members indicates the presence of one or more analytes of interest in the sample.
- detemining presence of the analyte or analytes of interest in the test sample is indicated by the substantial absence of bound detectable specific binding reagent aggregates at the second end of the porous member which were excluded from the porous member at first end of the porous member.
- the one or more detectable specific binding reagents are distinguished based on, for example, the color of different labels or the fluorescence of different labels designated for the detection and correlating to a specific analyte of interest, then the identity of each of the one or more analytes of interest suspected of being present in the test sample are revealed.
- substantially no detectable specific binding reagents analyte complexes are formed and hence are not excluded from the porous member, which allows any unbound detectable specific binding reagents to pass freely through the porous member.
- coagulation, agglutination, precipitation, accumulation, clumping, or plug formation by any means or as appropriate, increasing viscosity can be uses as part of the detection scheme.
- additives may be included in the present invention for specific purposes, such as for example, buffers, enzyme inhibitors, enzyme substrate or cofactors, preservatives, stabilizers, detergents, sugars, facilitators, activators, oxidants, reductants, or any other additives desired for a particular purpose.
- an absorbent pad in fluid communication with the first end, the second end, or both ends of the porous member of the present invention.
- the absorbent pad can be made of any material capable of holding an aqueous or liquid sample in fluid communication with the porous member such that the sample can flow evenly through the porous member by capillary action.
- the housing may be made of any rigid material capable of housing the components of the present invention
- the housing may be preferably made of an impermeable material that contains the liquid sample and the components of the present invention inside the device without allowing for any leaks for permeation.
- the housing may be made of, for example, plastic, glass, metal, rubber, or any other non-permeable material.
- the housing may preferably include one or more apertures.
- the housing may include, for example, an aperture located at the first end of the porous member to act as an inlet for introduction of the test sample to the porous member.
- the housing may also include an aperture located on the area of the control zone, located near the second end of the porous member to allow for detection of whether or not the test has reached completion.
- This Example provides a device detection of methycillin resistant Staphylococcus aureaus (“MRSA”).
- MRSA methycillin resistant Staphylococcus aureaus
- Pastorex Staph Plus Biorad, part #65356
- a slurry of MRSA was run on the slide agglutination assay per the manufacturer's instructions. Moderate agglutination was observed in the well containing the mixture of test latex and MRSA, and no agglutination was observed in the well containing a mixture of MRSA and the control latex.
- the suspension of red latex test particle was applied to several types of media including nitrocellulose and porous polyethylene.
- a porous polyethylene membrane (Porex, part #181071) with suitable chromatographic properties was selected.
- untreated particles were applied to this membrane, the pore space volume was uniformly filled with particles producing a dark red color throughout the membrane.
- An 18 millimeter wide ribbon of this membrane was laminated to a clear lexan support with double sided tape and cut into 6 millimeter wide test strips.
- test particles and 25 microliters of control latex particles were dispensed into separate new tubes.
- 0.5 microliters of yellow food coloring to impart contrasting color to the clear solution 5 microliters of heat killed MRSA was added to each tube, mixed and allowed to incubate for 1 minute at room temperature. Twelve microliters of either test or control mixture was added to the proximal ends of separate membrane strips and the strips were rapidly filled by capillary action.
- the control latex was not excluded from the membrane and a dark red/orange color was clearly visible at the end of the strip distal to the area of sample application.
- the test latex which had aggregated, was excluded from entering the membrane and only the yellow contrasting color of the solution was visible at the distal end of the strip.
- This Example provides a device detection of Streptococcus agalactiae (“Strep A”). Construction of the agglutination separation strip:
- the present invention includes an optional absorbent 11 pad at one end in fluid communication with the first porous member 12 .
- the first porous member is in fluid communication with the second porous member 14 , forming a junction 13 .
- An important aspect of the first porous member 12 of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the first porous member 12 only when the detectable specific binding reagent is not bound to any analyte.
- the present invention may optionally include an absorbent pad 14 in fluid communication with the second porous member 14 .
- the present invention may include a third porous member 16 , which includes the detectable specific binding reagents and is used to contact the test device in order to run the test.
- the present invention includes a porous member 16 , which has the ability to substantially allow any detectable specific binding reagent to pass freely through the porous member 16 only when the detectable specific binding reagent is not bound to any analyte and substantially arrest or impede the flow of any bound detectable specific binding reagent from the porous member 16 , especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents.
- This Example refers to FIG. 2A and FIG. 2B , and provides a device detection of human chorionic gonadotropin (“hCG”).
- hCG human chorionic gonadotropin
- the hCG (human chorionic gonadotropin) system is an excellent candidate for development of the agglutination process due to the characteristics of hormones in urine.
- Hormone levels are meaningful at very low concentrations, so detection of 10-60 mIUs needs to be effective.
- test strips were constructed as shown above, and stored in a dry container.
- test reagents including:
- test runs were performed by mixing the liquid latex-antibody solution mixed with the target antigen in a test tube, for zero to 10 minute incubations:
- This Example refers to FIG. 3A and FIG. 3B , and provides a device for dual antigen detection assay.
- the card based assay is based on the standard ICT card assay design with an additional test strip configured in the opposite direction ( FIG. 3A ):
- the cassette based assay design has the configuration of the test strips resembling that of ( FIG. 3B ):
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
- The present invention relates generally to the field of agglutination assays, and particularly to chromatographic exclusion assays and methods of use thereof.
- A variety of air agglutination assays are commercially available. These agglutination assays can be used for a variety of purposes, such as diagnosing conditions of disease or monitor treatments by analyzing trace analytes of interest in a sample. Certain agglutination assays are fully performed in solution, with the presence of visible analyte induced aggregates in the solution as indication of positive result. Other agglutination assays are performed utilizing a flow-through system where a reaction sample is passed through a small hole in order to concentrate analyte induced aggregates. Other agglutination assays are performed on dry porous strips, with the presence of anlalyte induced aggregates on the strip as indication of positive result.
- Many of the available agglutination assays suffer from lack of sensitivity and are prone to providing false-positive results. The agglutination assays that utilize a dry porous strip are especially prone to providing false-positive results because of difficulties in observing and detecting true agglutinates that are aggregated in or on the porous strip from random clumping of the agglutination assay components. Most agglutination assays, especially the flow-through agglutination assays, are prone to providing false-positive results because of the presence of non-specific particulate matter in a sample under test. The above described agglutination assays usually require a highly skilled person to determine the end result accurately as positive or negative, or to make the decision to run the test again for a more definitive result. While there has been a trend to produce more sensitive agglutination assays, further improvements in the sensitivity, effectiveness, and simplicity for home or clinic use of agglutination assays are desirable, and the present invention addresses the existing problems and provides related benefits.
- The present invention relates to agglutination assays and in particular to chromatographic exclusion assays and methods of use thereof. The present invention includes a novel strategy for determining the presence of one or more analytes on interest in a test sample by using chromatographic exclusion or separation of aggregates of bound detectable specific binding reagents that are accumulated at a particular and non-random location on the test device. In the absence of analytes of interest in the sample under test, the specific detectable binding reagent aggregates are not formed, and hence not excluded from the chromatographic media creating a distinct and readily differentiating event. However, it is also possible to separate the aggregates from single particles by retaining the non-aggregated particles, as in size-exclusion chromatography. The present invention is particularly adaptable as a simple test device for detection of diseases or monitoring of treatments at a doctor's office or in the home.
- The present invention recognizes that agglutination assays can be made more sensitive, more efficient, and more simple using chromatographic exclusion or separation of aggregates formed by the binding of a detectable specific binding reagent with the analytes of interest. Preferably, the chromatographically excluded or separated aggregates of a positive test result lead to a distinct event, such as the formation of a visible band on the chromatographic media, which is easily differentiated from the result of a negative test where no aggregates are excluded or separated from the chromatographic media.
- A first aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a test sample including a first porous member that includes one or more specific binding reagents that are capable of being detected and capable of binding to the analyte or analytes of interest suspected of being present in a test sample. The present invention also includes a second porous member that is in fluid communication with the first porous member, and is capable of substantially allowing any unbound detectable specific binding reagent to pass through freely, but substantially retaining any bound detectable specific binding reagent. When the sample is introduced to the first porous member of the present invention, the sample moves through the first and second porous member by capillary flow and the presence of the analyte or analytes of interest in the test sample is indicated by the presence of bound detectable specific binding reagent aggregates at the interface of the first porous member with the second porous member.
- A second aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a sample including a first porous member and a second porous member in fluid communication with one another forming a detection zone at the junction of the first and second porous members. A reaction zone positioned on the first porous member includes one or more detectable specific binding reagents that are able to bind to the analyte or analytes of interest suspected of being present in the sample under test and form aggregates. The one or more detectable specific binding reagents present at the reaction zone are capable of solublizing and moving to the second porous member by capillary flow. The second porous member is capable of substantially allowing any unbound detectable specific binding reagent to pass through freely, but substantially impeding the flow of any bound and aggregated detectable specific binding reagent. When the sample is introduced to the first porous member of the present invention, the sample moves through the first and second porous member by capillary flow and the presence of the analyte or analytes of interest in the test sample is indicated by the formation of an accumulation of bound detectable specific binding reagent aggregates at the junction of the first porous member with the second porous member, and the absence of the analyte or analytes of interest in the test sample is indicated by the absence or lack of an accumulation of bound detectable specific binding reagent aggregates at the junction of the first porous member with the second porous member.
- A third aspect of the present invention includes a method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of: providing a first porous member that includes one or more detectable specific binding reagents that are able to bind to the analyte or analytes of interest suspected of being present in the test sample; providing a second porous member, which is in fluid communication with the first porous member and forming a junction, such that the second porous member substantially allows any unbound detectable binding reagent to pass through freely, but substantially prevents the flow of any bound detectable specific binding reagent; introducing the test sample to the first porous member, such that the test sample moves through the first and second porous members by capillary flow and mobilizes the one or more detectable specific binding reagents of the first porous member; wherein the presence of the analyte or analytes of interest is indicated by the presence of bound detectable specific binding reagent aggregates at the junction of the first porous member with the second porous member.
- A fourth aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a sample including one or more detectable specific binding reagents capable of being detected and capable of binding to the one or more analytes of interest when added to the sample. The present invention also includes a first porous member and a second porous member in fluid communication with one another wherein the second porous member is capable of substantially allowing the one or more detectable specific binding reagents to pass through freely, but substantially retaining the detectable specific binding reagents when they are bound to an analyte or analytes of interest. Wherein the one or more detectable specific binding reagents are introduced to the sample forming a mixture and the mixture is then introduced to the first porous member, moving through the first and second porous members via capillary action, and the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagent aggregates at the interface of the first porous member with the second porous member.
- A fifth aspect of the present invention includes a method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of: mixing one or more detectable specific binding reagents able to bind to said one or more analytes of interest in the sample with the sample in order to form a mixture; providing a first porous member; providing a second porous member in fluid communication with the first porous member and forming a junction. The second porous member being capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the second porous member, but substantially preventing the flow of the one or more detectable specific binding reagents through the second porous member when bound to the analyte or analytes of interest; introducing the mixture to the first porous member, wherein the mixture moves by capillary action through the first and the second porous members; wherein, the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagents aggregates at the junction of the first and second porous members.
- A sixth aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a sample including a first porous member including one or more detectable specific binding reagents capable of being detected and capable of binding to the one or more analytes of interest when added to the sample. The present invention also includes a second and a third porous members wherein the third porous member is in fluid communication with the second porous member, and the third porous member is capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the third porous member, but substantially retaining the one or more detectable specific binding reagents when the one or more detectable specific binding reagents are bound to the one or more analytes of interest; whereby, the sample is introduced to the first porous member and the first porous member is brought into fluid contact with the second porous member, wherein the sample moves through the first, second, and third porous members by capillary flow and, wherein the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagents aggregates at the interface of the second porous member with the third porous member.
- A seventh aspect of the present invention includes a method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of: providing a first porous member; providing a second porous member; providing a third porous member in fluid communication with the second porous member forming a junction, and the third porous member being capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the third porous member, but substantially preventing the flow of the one or more detectable specific binding reagents through the third porous member when bound to the one or more analytes of interest; mixing one or more detectable specific binding reagents able to bind to the one or more analytes of interest in the sample with the sample forming a mixture; introducing the mixture to the first porous member; contacting the first porous member with the second porous member wherein the mixture moves by capillary action through the first, second, and third porous members; wherein, the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagents aggregates at the junction of the second and third porous members.
- An eighth aspect of the present invention includes a device for detecting the presence or absence of one or more analytes of interest in a test sample including one or more detectable specific binding reagents that are capable of being detected and capable of binding to the analyte or analytes of interest suspected of being present in a test sample. The present invention also includes a porous member having a first end and a second end, and is capable of substantially allowing any unbound detectable specific binding reagent to pass through freely, but substantially excluding any bound detectable specific binding reagent. When the sample is mixed with the one or more detectable binding agents and is introduced to the first end of the porous member of the present invention, the sample moves through the porous member by capillary flow and the presence of the analyte or analytes of interest in the test sample is indicated by the substantial absence of bound detectable specific binding reagent aggregates at the second end of the porous member which were excluded from the porous member at first end of the porous member.
- A ninth aspect of the present invention includes method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of: mixing one or more detectable specific binding reagents able to bind to said one or more analytes of interest in the sample with the sample in order to form a mixture; providing a porous member having a first end and a second end and capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the porous member, but substantially preventing the flow of the one or more detectable specific binding reagents through the porous member when they are bound to the analyte or analytes of interest; introducing the mixture to the first end of the porous member, wherein the mixture moves by capillary action through the porous member; wherein, the presence of the one or more analytes of interest is indicated by the substantial absence of the bound one or more detectable specific binding reagents aggregates at the second end of the porous member that were excluded from the porous member at the first end of the porous member.
- The designations “first”, “second”, or “third” as used throughout the description of the present invention are merely for purposes of convenience and are used in order to avoid any confusion. These terms are not used for and do not imply any special ordering or arrangements in terms of priority, element structure, reactivity, or the like.
-
FIG. 1A is a top view of an embodiment of the present invention. -
FIG. 2B is a top view of an embodiment of the present invention. - Introduction
- The present invention recognizes that agglutination assays can be made more sensitive, more efficient, and more simple using chromatographic exclusion or separation of aggregates formed by the binding of one or more detectable specific binding reagents with the analyte or analytes of interest at a particularly designated and non-random location on the test device. Preferably, the chromatographically excluded aggregates of a positive test result lead to a distinct event, such as the formation of a visible band on the chromatographic media, which is easily differentiated from the result of a negative test where no aggregates are excluded form the chromatographic media.
- As a non-limiting introduction to the breadth of the present invention, the present invention includes several general and useful aspects, including:
-
- 1) A device for detecting the presence or absence of one or more analytes of interest in a test sample including:
- a first porous member that includes one or more specific binding reagents that are capable of being detected and capable of binding to the analyte or analytes of interest suspected of being present in a test sample;
- a second porous member that is in fluid communication with the first porous member. The second porous member is capable of substantially allowing any unbound detectable specific binding reagent to pass through freely, but substantially retaining any bound detectable specific binding reagent;
- wherein, the sample is introduced to the first porous member and moves though the first and second porous members by capillary flow and the presence of the analyte or analytes of interest in the test sample is indicated by the presence of bound detectable specific binding reagent aggregates at the interface of the first porous member with the second porous member.
- 2) A device for detecting the presence or absence of one or more analytes of interest in a sample including:
- a first porous member and a second porous member in fluid communication with one another forming a detection zone at the junction of the first and second porous members;
- a reaction zone that is positioned on the first porous member that includes one or more detectable specific binding reagent that is able to bind to the analyte or analytes of interest suspected of being present in the sample under test. The one or more detectable specific binding reagents present at the reaction zone are capable of solublizing and moving to the second porous member by capillary flow. The second porous member is capable of substantially allowing any unbound detectable specific binding reagent to pass through freely, but substantially impeding the flow of any bound detectable specific binding reagent;
- wherein, the sample is introduced to the first porous member and moves though the first and second porous members by capillary flow and the presence of the analyte of interest in the test sample is indicated by the formation of an accumulation of bound detectable specific binding reagent aggregates at the junction of the first porous member with the second porous member, and the absence of the analyte of interest in the test sample is indicated by the absence of an accumulation of bound detectable specific binding reagent aggregates at the junction of the first porous member with the second porous member.
- 3) A method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of:
- a) providing a first porous member that includes one or more detectable specific binding reagents that are able to bind to the analyte or analytes of interest suspected of being present in the test sample;
- b) providing a second porous member, which is in fluid communication with the first porous member forming a junction, such that the second porous member substantially allows any unbound detectable binding reagent to pass through freely, but substantially prevents the flow of any bound detectable specific binding reagent;
- c) introducing the test sample to the first porous member, such that the test sample moves through the first and second porous members by capillary flow and mobilizes the one or more detectable specific binding reagent of the first porous member;
- wherein the presence of the analyte or analytes of interest is indicated by the presence of bound detectable specific binding reagent aggregates at the junction of the first porous member with the second porous member.
- 4) A device for detecting the presence or absence of one or more analytes of interest in a sample including:
- one or more detectable specific binding reagents capable of being detected and capable of binding to the one or more analytes of interest when added to the sample;
- a first porous member;
- a second porous member in fluid communication with the first porous member, the second porous member being capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the second porous member, but substantially retaining the one or more detectable specific binding reagents when the one or more detectable specific binding reagents are bound to the analytes of interest;
- wherein, the one or more detectable specific binding reagents are introduced to the sample forming a mixture, and said mixture is introduced to said first porous member and moves through the first and second porous member by capillary flow, and the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagent aggregates at the interface of the first porous member with the second porous member.
- 5) A method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of:
- a) mixing one or more detectable specific binding reagents able to bind to the one or more analytes of interest in the sample with the sample forming a mixture;
- b) providing a first porous member;
- c) providing a second porous member in fluid communication with the first porous member forming a junction, the second porous member being capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the second porous member, but substantially preventing the flow of the one or more detectable specific binding reagents through the second porous member when bound to the one or more analytes of interest;
- d) introducing the mixture to the first porous member, wherein the mixture moves by capillary action through the first and the second porous members;
- wherein, the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagent aggregates at the junction of the first and second porous members.
- 6) A device for detecting the presence or absence of one or more analytes of interest in a sample including:
- a first porous member including one or more detectable specific binding reagents capable of being detected and capable of binding to the one or more analytes of interest when added to the sample;
- a second porous member;
- a third porous member in fluid communication with the second porous member, the third porous member being capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the third porous member, but substantially retaining the one or more detectable specific binding reagents when the one or more detectable specific binding reagents are bound to the one or more analytes of interest;
- whereby, the sample is introduced to the first porous member and the first porous member is brought into fluid contact with the second porous member, wherein the sample moves through the first, second, and third porous members by capillary flow and, wherein the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagent aggregates at the interface of the second porous member with the third porous member.
- 7) A method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of:
- a) providing a first porous member;
- b) providing a second porous member;
- c) providing a third porous member in fluid communication with the second porous member forming a junction, the third porous member substantially allowing the one or more detectable specific binding reagents to pass freely through the third porous member, but substantially preventing the flow of the one or more detectable specific binding reagents through the third porous member when bound to the one or more analytes of interest;
- d) mixing one or more detectable specific binding reagents able to bind to the one or more analytes of interest in the sample with the sample forming a mixture;
- e) introducing the mixture to the first porous member;
- f) contacting the first porous member with the second porous member wherein the mixture moves by capillary action through the first, second, and third porous members;
- wherein, the presence of the one or more analytes of interest is indicated by the presence of the bound one or more detectable specific binding reagent aggregates at the junction of the second and third porous members.
- 8) A device for detecting the presence or absence of one or more analytes of interest in a sample including:
- one or more detectable specific binding reagents capable of being detected and capable of binding to the one or more analytes of interest when added to the sample;
- a porous member having a first end and a second end, the porous member being capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the porous member, but substantially excluding the one or more detectable specific binding reagents when the one or more detectable specific binding reagents are bound to the analytes of interest;
- wherein, the one or more detectable specific binding reagents are introduced to the sample forming a mixture and the mixture is introduced to the first end of the porous member and moves through the porous member by capillary flow and the presence of the one or more analytes of interest is indicated by the substantial absence of the bound one or more detectable specific binding reagents aggregates at the second end of the porous member that were excluded from the porous member at the first end.
- 9) A method of detecting the presence or absence of one or more analytes of interest in a sample including the steps of:
- a) mixing one or more detectable specific binding reagents able to bind to the one or more analytes of interest in the sample with the sample forming a mixture;
- b) providing a porous member having a first end and a second end being capable of substantially allowing the one or more detectable specific binding reagents to pass freely through the porous member, but substantially excluding the one or more detectable specific binding reagents through the porous member when bound to the one or more analytes of interest;
- d) introducing the mixture to the first end of the porous member, wherein the mixture moves by capillary action through the porous member;
- wherein, the presence of the one or more analytes of interest is indicated by the substantial absence of the bound one or more detectable specific binding reagent aggregates at the second end of the porous members that were excluded from the porous member at the first end.
- 1) A device for detecting the presence or absence of one or more analytes of interest in a test sample including:
- The present invention relates to agglutination assays and in particular to chromatographic exclusion assays and methods of use thereof The present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion or separation of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device. The particular arrangement of the chromatographic media of the present invention is utilized in order to increase sensitivity and eliminate subjective interpretation of the test result. The present invention provides an exclusion of aggregate that is definitively detectable when the analyte or analytes of interest are present in the sample, and in the absence of the analyte or analytes of interest in the sample under test, the detectable specific binding reagent aggregates are not formed at the detection zone of the test device, and hence, are not excluded from the chromatographic media, which creates a distinct and readily differentiating event. The present invention is particularly adaptable as a simple and rapid test device for detection of diseases or monitoring of treatments at a doctor's office or in the home.
- Further objectives and advantages of the present invention will become apparent as the description proceeds and when taken in conjunction with the accompanying drawings. To gain a full appreciation of the scope of the present invention, it will be further recognized that various aspects of the present invention can be combined to make desirable embodiments of the invention.
- Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the manufacture or laboratory procedures described below are well known and commonly employed in the art. The technical terms used herein have their ordinary meaning in the art that they are used, as exemplified by a variety of technical dictionaries. Where a term is provided in the singular, the inventor also contemplates the plural of that term. The nomenclature used herein and the procedures described below are those well known and commonly employed in the art.
- Description of the Porous Members in Fluid Communication Forming a Junction
- The present invention includes a first porous member, which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample. Such material include, but are not limited to, glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- An important aspect of the first porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the first porous member, whether the detectable specific binding reagents are bound to analytes of interest or not Furthermore the pores or channels of the first porous member should be large enough as to not substantially impede or retard the free flow of any coagulated or aggregates of bound detectable specific binding reagent through the first porous member. The first porous member may have pores or channels of any size, which will be largely dependent on the size of the specific binding reagent being used or any particle or label which may be used as a label. The pore size of the first porous member may be, for example, about 10 micrometers to about 500 micrometer, and preferably about 50 micrometers to about 200 micrometers.
- The present invention also includes a second porous member that is in fluid communication with the first porous member of the present invention. Preferably, one end of the first porous member is in contact and joins one end of the second porous member, forming a junction where the first porous member ends and the second porous member begins in fluid contact. Any aqueous or liquid sample that contacts the first porous member will be moved by capillary action towards the second porous member where it moves past the junction of the first and second porous members and continues to moves through the second porous member. The second porous member of the present invention can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample. Such material include, but are not limited to, glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- An important aspect of the second porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the second porous member only when the detectable specific binding reagent is not bound to any analyte. If the one or more analytes of interest are present in the test sample, binding events occur between the one or more detectable specific binding reagents of the present invention and the one or more analytes of interest of the test sample and complexes of detectable specific binding reagents and analytes are formed. Preferably, the second porous member substantially arrests or impedes the flow of any bound detectable specific binding reagent, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of detectable specific binding reagents complexes. The second porous member may have pores or channels of any size, which will be largely dependent on the size of the specific binding reagent being used or any particle or label which may be used as a label. Because the second porous member must substantially allow unbound detectable specific binding reagents to flow freely through the second porous member, the pore size of the second porous member may be, for example, about 0.1 micrometers to about 100 micrometers, and preferably about 1 micrometer to about 20 micrometers.
- The capillary action provides the driving source or pumping force of the movement of the liquid through the device. The device is normally utilized in the horizontal position so that the capillary flow of the liquid sample through the device is normally a lateral flow, which is not affected by the force of gravity. However, certain embodiments of the present invention may include added driving forces, such as for example, gravity or certrifugal force.
- Description of the Detectable Specific Binding Reagents of the Present Invention
- The detectable specific binding reagents of the present invention are preferably substantially freely mobile when in the solublized or moist state. Preferably each type of detectable specific binding regent of the first porous member is capable of binding a single analyte of interest suspected of being present in the test sample forming aggregates, and each type of detectable specific binding reagent is distinguishable from one another. The detectable specific binding reagents can be any anti-analyte, namely a compound that is capable of specifically binding to an analyte of interest forming aggregates. The analytes of interest may include, for example, any material capable of being detected by a specific binding reagent including any cells or viruses or any components thereof. The analytes of interest may also include any small organic molecules, such as drugs, hormones, steroids, neurotransmitters, growth factors, metabolites, or other chemicals. The analytes of interest may include, for example, any large organic molecules, such as nucleic acids, proteins, polysaccharides, or other large molecules.
- The detectable specific binding reagent of the present invention may be, for example, an intact antibody, such as a polyclonal or a monoclonal antibody. Preferably, the detectable specific binding regent is an antibody, for example, human or other mammalian IgG, IgG1 IgG2, IgG3, IgG4, IgM, IgA, IgY, SigA, or IgE. The detectable specific binding reagent of the present invention may also be an antibody fragment, such as an antigen-binding fragment or Fab, or an F(ab′)2 fragment, or the antigen-binding site of an antibody (such as a complementarity-determining region of an antibody).
- In some embodiments, the detectable specific binding reagent is preferably a purified, high affinity monoclonal antibody that specifically binds an analyte of interest suspected of being present in the test sample. The detectable specific binding reagent may, in other embodiments, be an antigen, a ligand, or a receptor. The detectable specific binding reagent may bind to more than one type of analyte, but preferably binds only one type of analyte that is suspected of being present in the test sample. The detectable specific binding reagent may be comprised of more than one reagent; for example, the detectable specific binding reagent may comprise more than one type of monoclonal antibody or antibody fragment, each of which specifically binds the same type of analyte of interest suspected of being present in the test sample. The detectable specific binding reagent of the present invention can include, without limitation, peptides, polypeptides, antibodies, Fab fragments, fusion proteins, chimeric or hybrid molecules, nucleic acids, nucleic acid mimics (for example, peptide nucleic acids), carbohydrates, cell surface antigens, receptors, ligands, or combinations thereof Preferably, the detectable specific binding reagent includes an antibody (monoclonal or polyclonal, natural, modified, or recombinant) or an antibody fragment (such as an Fab fragment or single-chain antibody variable region fragment). Methods of preparing, modifying, and using such antibodies or antibody fragments are known in the art (see, for example, “Antibodies: A Laboratory Manual”, E. Harlow and D. Lane, editors, Cold Spring Harbor Laboratory, 1988, 726 pp; “Monoclonal Antibodies: A Practical Approach”, P. Shepherd and C. Dean, editors, Oxford University Press, 2000, 479 pp.; and “Chicken Egg Yolk Antibodies, Production and Application: IgY-Technology (Springer Lab Manual)”, by R Schade et al., editors, Springer-Verlag, 2001, 255 pp., which are incorporated by reference in their entirety herein).
- The detectable specific binding reagent can include an antigen, such as an antigen capable of specifically binding to an antibody that recognizes an analyte of interest. The detectable binding reagent can include a nucleic acid or nucleic acid mimic aptamer that binds a target such as a peptide or small molecule, or a receptor that binds a ligand, or a ligand that binds a receptor. The detectable specific binding reagent may be capable of binding to a mimotope, such as a peptide, that mimics an analyte of interest (see, for example, Kieber-Emmons (1998) Immunol. Res., 17:95-108; Shin et al. (2001) Infect. Immun., 69:3335-3342; Beenhouwer et al. (2002) J. Immunol., 169:6992-6999; Hou and Gu (2003) J. Immunol., 170:4373-4379; and Tang et al. (2003) Clin. Diagn. Lab. Immunol., 10:1078-1084, which are incorporated by reference in their entirety herein).
- The detectable specific binding reagent also includes the capacity to be detected and preferably each type of detectable specific binding reagent is distinguishably detectable from one another. The detectable specific binding reagents' capacity to be detected may be any quality capable of being detected, and includes, for example, detection by a label such as a color label including dyes, metal sols, and latex particles, or capacity to be detected by a label such as, for example, fluorescent label, radioactive label, magnetic label or chemiluminescent label. The detectable specific binding reagents' capacity to be detected may also be, for example, detection by size, charge, polarity, hydrophobicity, hydrophilicity, lipophilicity, or viscosity. In order to identify one or more analytes of interest in a test sample, each detectable specific binding reagent that is specific for a certain analyte of interest may include a different capacity to be detected, for example, different color labels, different fluorescent labels, or any differentiating capacity to be distinguished, or any combination thereof.
- Depending on the detectable specific binding reagents' chosen capacity to be detected, different detection methods may be employed in different protocols. Detection methods have enormous diversity and are presently available, and any detection method may be employed depending on the protocol. The detection methods may include, for example, direct detection of labels such as metal sols, colored labels, colored beads, colored particles, or fluorescent labels, either by way of direct vision or by way of a microscope. Labels of the present invention may be of any size and it will be largely dependent on the test that is to be performed and the pore size or chanel size of the porous members of the present invention. The labels of the present invention may have a size range of, for example, about 0.01 micrometers in diameter to about 50 micrometers in diameter, and preferably about 0.02 micrometers in diameter to about 2 micrometers in diameter. The detection methods may also include, for example, indirect detection of detectable labels such as magnetic labels, radioactive labels, or the measurement of scattered light to measure a change in the size population.
- I. Device and Method for Detecting the Presence or Absence of One or More Analytes of Interest in a Test Sample (Internal Mixture)
- The present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device.
- The present invention includes a first porous member including one or more detectable specific binding reagents that are each capable of being detected and each capable of biding to an analyte of interest in the test sample. The detectable specific binding reagents of the first porous members are preferably dried onto or otherwise temporarily immobilized into a reaction zone or region of the first porous member such that the reaction zone of the first porous member is impregnated by the dried detectable specific binding reagents. The impregnated detectable specific binding reagents of the first porous member are preferably substantially freely mobile when in the moist or solublized state.
- An important aspect of the first porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the first porous member, whether the detectable specific binding reagents are bound to analytes of interest or not. Furthermore the pores of the first porous member should be large enough as to not substantially impede the free flow of any coagulated or aggregates of bound detectable specific binding reagent through the first porous member.
- The present invention includes a second porous member that is in fluid communication with the first porous member of the present invention. Preferably, one end of the first porous member is in contact and joins one end of the second porous member, forming a junction where the first porous member ends and the second porous member begins in fluid contact. Any aqueous or liquid sample that contacts the first porous member will be moved by capillary action towards the second porous member where it moves past the junction of the first and second porous members and continues to moves through the second porous member.
- An important aspect of the second porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the second porous member only when the detectable specific binding reagent is not bound to any analyte. Preferably, the second porous member substantially retains and impedes the flow of any bound detectable specific binding reagent, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents.
- The presence or absence of one or more analytes in a test sample is determined by contacting the test sample to the first porous member of the present invention. The test sample moves through the first porous member by capillary flow where the one or more detectable specific binding reagents of the first porous member are solubilized and are mixed in with the test sample forming a mixture. If one or more analytes of interest are present in the test sample, the one or more detectable specific binding reagents bind the one or more analytes of interest forming a complex, and the detectable specific binding reagents analyte complexes move freely through the first porous member by capillary flow towards the second porous member of the present invention. At the junction of the first and second porous members, if the one or more analytes of interest are present in the test sample, the bound detectable specific binding reagents analyte complexes are substantially retained by the second porous member, which does not substantially allow bound detectable specific binding reagents analyte complexes to pass freely through the second porous member. Determining the presence of aggregated bound detectable specific binding reagents analyte complexes that are substantially accumulated at the junction of the first and second porous members indicates the presence of one or more analytes of interest in the sample. Furthermore, if the one or more detectable specific binding reagents are distinguished based on, for example, the color of different labels or the fluorescence of different labels designated for the detection and correlating to a specific analyte of interest, then the identity of each of the one or more analytes of interest suspected of being present in the test sample are revealed. Alternatively, if the one or more analytes of interest are not present in the test sample, substantially no detectable specific binding reagents analyte complexes are formed and hence are not retained by the second porous member, which allows any unbound detectable specific binding reagents to pass freely through the second porous member. Besides coagulation, agglutination, precipitation, accumulation, clumping, or plug formation, by any means or as appropriate, increasing viscosity can be used as part of the detection scheme.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example, a control zone located on the second porous member in order to ensure a test has been completed. Preferably, the control zone is located on the second porous member at some distance away from the junction of the first and second porous members. The control zone may include a region on the second porous member at some distance away from the junction of the first and second porous members, where it can indicate that the test sample has travelled past the detection zone or the junction of the first and second porous members. A control compound can be used, for example a colored solution, which can be mixed in with the test sample and be detected at the control zone indicating that the test sample has travelled through the device past the junction of the first and second porous members. The control compound may also be, for example a control detectable label such as control latex. The control zone can include immobilized specific biding reagents that are capable of detecting a control compound. The control compound, may include for example, a known substance that does not bind to the detectable specific binding regents of the first porous member. The control compound can be mixed in with the sample, which will pass through the first and second porous members without being retained at the junction of the first and second porous member and reach the control zone where it will bind to the immobilized specific binding reagent of the control zone, indicating that the test sample has travelled through the test device and has passed the junction of the first and second porous members.
- In addition to the components of the present invention described above, other additives may be included in the present invention for specific purposes, such as for example, buffers, enzyme inhibitors, enzyme substrate or cofactors, preservatives, stabilizers, solublizing agents, detergents, sugars, facilitators, activators, oxidants, reductants, or any other additives desired for a particular purpose.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example, an absorbent pad in fluid communication with the first porous member of the present invention. The absorbent pad can be made of any material capable of holding an aqueous or liquid sample in fluid communication with the first porous member such that the sample can flow evenly through the first porous member by capillary action. The present invention may also include an absorbent pad in fluid communication with the distal end of the second porous member, having sufficient capacity to absorb any liquid and acting as a distal sink at the end of the test device in order to remove any excess liquid sample from the components of the present invention.
- The present invention may also include, for example, a filter positioned between the first porous member and the second porous member and in a continuous fluid communication with the first and second porous members. The filter may act to filter out or trap larger components above a certain desired range, which may be present in the test sample. For example, if the test sample includes a blood sample, the filter may be used to filter out blood cells or other components of blood larger than a desired size.
- The present invention is adaptable to be used with a plurality of similar first porous member and second porous member in fluid communication as described above. These plurality of porous members may be used, for example to run different desired tests on the same test sample at the same time. These plurality of porous members may be used in parallel configuration or stacked on top of one another.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example a housing. The housing may be made of any rigid material capable of housing the components of the present invention The housing may be preferably made of an impermeable material that contains the liquid sample and the components of the present invention inside the device without allowing for any leaks for permeation. The housing may be made of, for example, plastic, glass, metal, rubber, or any other non-permeable material. The housing may preferably include one or more apertures. The housing may include, for example, an aperture located on the area of the first porous member to act as an inlet for introduction of the test sample to the first porous member, located at some distance away from the junction of the first and second porous member. The housing may also include an aperture located on the area of the detection zone or the junction of the first and second porous members to allow for detection of whether the test result turned positive or negative. The housing may also include an aperture located on the area of the control zone, located on the second porous member at some distance away from the detection zone or the junction of the first and second porous members to allow for detection of whether or not the test has reached completion.
- II. Device and Method for Detecting the Presence or Absence of One or More Analytes of Interest in a Test Sample (External Mixture)
- The present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device.
- The present invention includes one or more detectable specific binding reagents that are each capable of being detected and each capable of binding to an analyte of interest in the test sample when added to the test sample. The detectable specific binding reagents of the present invention may be separately mixed with the test sample suspected of containing one or more analytes of interest forming a mixture, and incubated for a time period sufficient to allow the one or more detectable specific binding reagents to bind the one or more analytes of interest in the test sample. After proper incubation, the mixture of test sample and detectable specific binding reagents is used to run through the test device of the present invention.
- The present invention includes a first porous member, which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- An important aspect of the first porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the first porous member, whether the detectable specific binding reagents are bound to analytes of interest or not. Furthermore the pores of the first porous member should be large enough as to not substantially impede the free flow of any coagulated or aggregates of bound detectable specific binding reagent through the first porous member.
- The present invention includes a second porous member that is in fluid communication with the first porous member of the present invention. Preferably, one end of the first porous member is in contact and joins one end of the second porous member, forming a junction where the first porous member ends and the second porous member begins in fluid contact. Any aqueous or liquid sample that contacts the first porous member will be moved by capillary action towards the second porous member where it moves pass the junction of the first and second porous members and continues to moves through the second porous member.
- An important aspect of the second porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the second porous member only when the detectable specific binding reagent is not bound to any analyte. Preferably, the second porous member substantially retains and impedes the flow of any bound detectable specific binding reagent, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents.
- A test sample may be tested by separately mixing the test sample suspected of containing one or more analytes of interest with the detectable specific binding reagents of the present invention forming a mixture, and incubated for a time period sufficient to allow the one or more detectable specific binding reagents to bind the one or more analytes of interest in the test sample. The presence or absence of one or more analytes in a test sample is determined by contacting the test sample and detectable specific binding reagent mixture to the first porous member of the present invention. If one or more analytes of interest are present in the test sample, the one or more detectable specific binding reagents bind the one or more analytes of interest forming a complex in the test mixture, and the detectable specific binding reagents analyte complexes move freely through the first porous member by capillary flow towards the second porous member of the present invention. At the junction of the first and second porous members, if the one or more analytes of interest are present in the test sample, the bound detectable specific binding reagents analyte complexes are substantially retained by the second porous member, which does not substantially allow bound detectable specific binding reagents analyte complexes to pass freely through the second porous member. Determining the presence of aggregated bound detectable specific binding reagents analyte complexes that are substantially accumulated at the junction of the first and second porous members indicates the presence of one or more analytes of interest in the sample. Furthermore, if the one or more detectable specific binding reagents are distinguished based on, for example, the color of different labels or the fluorescence of different labels designated for the detection and correlating to a specific analyte of interest, then the identity of each of the one or more analytes of interest suspected of being present in the test sample are revealed. Alternatively, if the one or more analytes of interest are not present in the test sample, substantially no detectable specific binding reagents analyte complexes are formed and hence are not retained by the second porous member, which allows any unbound detectable specific binding reagents to pass freely through the second porous member. Besides coagulation, agglutination, precipitation, accumulation, clumping, or plug formation, by any means or as appropriate, increasing viscosity can be used as part of the detection scheme.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example, a control zone located on the second porous member in order to ensure a test has been completed. Preferable, the control zone is located on the second porous member at some distance away from the junction of the first and second porous members. The control zone may include a region on the second porous member at some distance away from the junction of the first and second porous members, where it can indicate that the test sample has travelled past the detection zone or the junction of the first and second porous members. A control compound can be used, for example a colored solution, which can be mixed in with the test sample and be detected at the control zone indicating that the test sample has travelled through the device past the junction of the first and second porous members. The control compound may also be, for example a control detectable label such as control latex. The control zone can include immobilized specific biding reagents that are capable of detecting a control compound. The control compound, may include for example, a known substance that does not bind to the detectable specific binding regents used to test the test sample. The control compound can be mixed in with the sample, which will pass through the first and second porous members without being retained at the junction of the first and second porous member and reach the control zone where it will bind to the immobilized specific binding reagent of the control zone, indicating that the test sample has travelled through the test device and has passed the junction of the first and second porous members.
- In addition to the components of the present invention described above, other additives may be included in the present invention for specific purposes, such as for example, buffers, enzyme inhibitors, enzyme substrate or cofactors, preservatives, stabilizers, solublizing agents, detergents, sugars, facilitators, activators, oxidants, reductants, or any other additives desired for a particular purpose.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example, an absorbent pad in fluid communication with the first porous member of the present invention. The absorbent pad can be made of any material capable of holding an aqueous or liquid sample in fluid communication with the first porous member such that the sample can flow evenly through the first porous member by capillary action. The present invention may also include an absorbent pad in fluid communication with the distal end of the second porous member, having sufficient capacity to absorb any liquid and acting as a distal sink at the end of the test device in order to remove any excess liquid sample from the components of the present invention.
- The present invention may also include, for example, a filter positioned between the first porous member and the second porous member and in a continuous fluid communication with the first and second porous members. The filter may act to filter out or trap larger components above a certain desired range, which may be present in the test sample. For example, if the test sample includes a blood sample, the filter may be used to filter out blood cells or other components of blood larger than a desired size.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example a housing. The housing may be made of any rigid material capable of housing the components of the present invention The housing may be preferably made of an impermeable material that contains the liquid sample and the components of the present invention inside the device without allowing for any leaks for permeation. The housing may be made of, for example, plastic, glass, metal, rubber, or any other non-permeable material. The housing may preferably include one or more apertures. The housing may include, for example, an aperture located on the area of the first porous member to act as an inlet for introduction of the test sample to the first porous member, located at some distance away from the junction of the first and second porous member. The housing may also include an aperture located on the area of the detection zone or the junction of the first and second porous members to allow for detection of whether the test result turned positive or negative. The housing may also include an aperture located on the area of the control zone, located on the second porous member at some distance away from the detection zone or the junction of the first and second porous members to allow for detection of whether or not the test has reached completion.
- III. Device and Method for Detecting the Presence or Absence of One or More Analytes of Interest in a Test Sample (Internal Mixture—3 Porous Members)
- The present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device. The analytes of interest may include any analyte capable of being detected.
- The present invention includes a first porous member, which can be made of and include any type of material, that is capable of retaining liquid and allowing the liquid to be transported away by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample. Such material include, but are not limited to, glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- The present invention includes a second porous member, which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample. Such material include glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- An important aspect of the second porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the first porous member, whether the detectable specific binding reagents are bound to analytes of interest or not. Furthermore the pores of the first porous member should be large enough as to not substantially impede the free flow of any coagulated or aggregates of bound detectable specific binding reagent through the first porous member.
- The present invention includes a third porous member that is in fluid communication with the second porous member of the present invention. Preferably, one end of the second porous member is in contact and joins one end of the third porous member, forming a junction where the second porous member ends and the third porous member begins in fluid contact. Any aqueous or liquid sample that contacts the second porous member will be moved by capillary action towards the third porous member where it moves past the junction of the second and third porous members and continues to moves through the third porous member. The third porous member of the present invention can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample.
- An important aspect of the third porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the third porous member only when the detectable specific binding reagent is not bound to any analyte. Preferably, the third porous member substantially retains and impedes the flow of any bound detectable specific binding reagent, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents.
- The presence or absence of one or more analytes in a test sample is determined by contacting the test sample to the first porous member of the present invention. The test sample is then retained by the first porous member where the one or more detectable specific binding reagents of the first porous member are solubilized and are mixed in with the test sample forming a mixture. If one or more analytes of interest are present in the test sample, the one or more detectable specific binding reagents bind the one or more analytes of interest forming a complex. When the first porous member of the present invention containing the test sample mixture is contacted with the second porous member of the present invention, detectable specific binding reagents analyte complexes move freely through from the first porous member to the second porous member by capillary flow towards the third porous member of the present invention. At the junction of the second and third porous members, if the one or more analytes of interest are present in the test sample, the bound detectable specific binding reagents analyte complexes are substantially retained by the third porous member, which does not substantially allow bound detectable specific binding reagents analyte complexes to pass freely through the third porous member. Determining the presence of aggregated bound detectable specific binding reagents analyte complexes that are substantially accumulated at the junction of the second and third porous members indicates the presence of one or more analytes of interest in the sample. Furthermore, if the one or more detectable specific binding reagents are distinguished based on, for example, the color of different labels or the fluorescence of different labels designated for the detection and correlating to a specific analyte of interest, then the identity of each of the one or more analytes of interest suspected of being present in the test sample are revealed. Alternatively, if the one or more analytes of interest are not present in the test sample, substantially no detectable specific binding reagents analyte complexes are formed and hence are not retained by the second porous member, which allows any unbound detectable specific binding reagents to pass freely through the second porous member. Besides coagulation, agglutination, precipitation, accumulation, clumping, or plug formation, by any means or as appropriate, increasing viscosity can be uses as part of the detection scheme.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example, a control zone located on the third porous member in order to ensure a test has been completed. Preferably, the control zone is located on the third porous member at some distance away from the junction of the second and third porous members. The control zone may include a region on the third porous member at some distance away from the junction of the second and third porous members, where it can indicate that the test sample has travelled past the detection zone or the junction of the second and third porous members. A control compound can be used, for example a colored solution, which can be mixed in with the test sample and be detected at the control zone indicating that the test sample has travelled through the device past the junction of the second and third porous members. The control compound may also be, for example a control detectable label such as control latex. The control zone can include immobilized specific biding reagents that are capable of detecting a control compound. The control compound, may include for example, a known substance that does not bind to the detectable specific binding regents of the first porous member. The control compound can be mixed in with the sample, which will pass from the first porous member to the second porous member upon contact and moves through the second and third porous members without being retained at the junction of the second and third porous member and reach the control zone where it will bind to the immobilized specific binding reagent of the control zone, indicating that the test sample has travelled through the test device and has passed the junction of the second and third porous members.
- In addition to the components of the present invention described above, other additives may be included in the present invention for specific purposes, such as for example, buffers, enzyme inhibitors, enzyme substrate or cofactors, preservatives, stabilizers, detergents, sugars, facilitators, activators, oxidants, reductants, or any other additives desired for a particular purpose.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example, an absorbent pad in fluid communication with the first porous member or the second porous member of the present invention. The absorbent pad can be made of any material capable of holding an aqueous or liquid sample in fluid communication with the first porous member or the second porous member. If the absorbent pad is positioned in fluid contact with the first porous member, the sample can be retained evenly through the first porous member and be transferred evenly to the second porous member upon contact and flow evenly through the first porous member by capillary action. If the absorbent pad is positioned in fluid contact with the second porous member, the sample can be flow evenly through the second porous member and into the second porous by capillary action. The present invention may also include an absorbent pad in fluid communication with the distal end of the third porous member, having sufficient capacity to absorb any liquid and acting as a distal sink at the end of the test device in order to remove any excess liquid sample from the components of the present invention.
- The present invention may also include, for example, a filter positioned between the second porous member and the third porous member and in a continuous fluid communication with the first and second porous members. The filter may act to filter out or trap larger components above a certain desired range, which may be present in the test sample. For example, if the test sample includes a blood sample, the filter may be used to filter out blood cells or other components of blood larger than a desired size.
- The present invention is adaptable to be used with a plurality of similar second porous member and third porous member in fluid communication as described above. These plurality of porous members may be used, for example to run different desired tests on the same test sample at the same time. These plurality of porous members may be used in parallel configuration or stacked on top of one another.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example a housing. The housing may be made of any rigid material capable of housing the components of the present invention The housing may be preferably made of an impermeable material that contains the liquid sample and the components of the present invention inside the device without allowing for any leaks for permeation. The housing may be made of, for example, plastic, glass, metal, rubber, or any other non-permeable material. The housing may preferably include one or more apertures. The housing may include, for example, an aperture located on the area of the second porous member to act as an inlet for introduction of the test sample to the second porous member, located at some distance away from the junction of the second and third porous member. The housing may also include an aperture located on the area of the detection zone or the junction of the second and third porous members to allow for detection of whether the test result turned positive or negative. The housing may also include an aperture located on the area of the control zone, located on the third porous member at some distance away from the detection zone or the junction of the second and third porous members to allow for detection of whether or not the test has reached completion.
- IV. Device and Method for Detecting the Presence or Absence of One or More Analytes of Interest in a Test Sample (External Mixture—Single Porous Member)
- The present invention includes a novel strategy for determining the presence of one or more analytes in a test sample by using chromatographic exclusion of aggregates formed by the binding of one or more detectable specific binding reagents at a defined zone of detection on the test device. The analytes of interest may include any analyte capable of being detected.
- The present invention includes one or more detectable specific binding reagents that are each capable of being detected and each capable of binding to an analyte of interest in the test sample when added to the test sample. The detectable specific binding reagents of the present invention may be separately mixed with the test sample suspected of containing one or more analytes of interest forming a mixture, and incubated for a time period sufficient to allow the one or more detectable specific binding reagents to bind the one or more analytes of interest in the test sample. After proper incubation, the mixture of test sample and detectable specific binding reagents is used to run through the, test device of the present invention.
- The present invention includes a porous member having a first end and a second end, which can be made of and include any type of material, that is capable of transporting a liquid sample by capillary action, or capillary flow, wicking, or simple wetting of a liquid sample. Such material include glass fiber, nitrocellulose, paper, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, and copolymers thereof, cellulose polymers, metals, or composites made up of a combination of these materials.
- The capillary action provides the driving source or pumping force of the movement of the liquid through the device. The device is normally utilized in the horizontal position so that the capillary flow of the liquid sample through the device is normally a lateral flow, which is not affected by the force of gravity.
- An important aspect of the porous member of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the porous member only when the detectable specific binding reagent is not bound to any analyte. Preferably, the porous member substantially excludes any bound detectable specific binding reagent from the porous member, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents.
- The capillary action provides the driving source or pumping force of the movement of the liquid through the device. The device is normally utilized in the horizontal position so that the capillary flow of the liquid sample through the device is normally a lateral flow, which is not affected by the force of gravity. However, certain embodiments of the present invention may include added driving forces, such as for example, gravity or certrifugal force.
- A test sample may be tested by separately mixing the test sample suspected of containing one or more analytes of interest with the detectable specific binding reagents of the present invention forming a mixture, and incubated for a time period sufficient to allow the one or more detectable specific binding reagents to bind the one or more analytes of interest in the test sample. The presence or absence of one or more analytes in a test sample is determined by contacting the test sample and detectable specific binding reagent mixture to the first end of the porous member of the present invention. If one or more analytes of interest are present in the test sample, the one or more detectable specific binding reagents bind the one or more analytes of interest forming a complex in the test mixture, and the detectable specific binding reagents analyte complexes are substantially excluded from the porous member, which does not substantially allow bound detectable specific binding reagents analyte complexes to pass freely through the second porous member. Determining the presence of aggregated bound detectable specific binding reagents analyte complexes that are substantially accumulated at the first end of the porous members indicates the presence of one or more analytes of interest in the sample. Alternatively, detemining presence of the analyte or analytes of interest in the test sample is indicated by the substantial absence of bound detectable specific binding reagent aggregates at the second end of the porous member which were excluded from the porous member at first end of the porous member. Furthermore, if the one or more detectable specific binding reagents are distinguished based on, for example, the color of different labels or the fluorescence of different labels designated for the detection and correlating to a specific analyte of interest, then the identity of each of the one or more analytes of interest suspected of being present in the test sample are revealed. Alternatively, if the one or more analytes of interest are not present in the test sample, substantially no detectable specific binding reagents analyte complexes are formed and hence are not excluded from the porous member, which allows any unbound detectable specific binding reagents to pass freely through the porous member. Besides coagulation, agglutination, precipitation, accumulation, clumping, or plug formation, by any means or as appropriate, increasing viscosity can be uses as part of the detection scheme.
- In addition to the components of the present invention described above, other additives may be included in the present invention for specific purposes, such as for example, buffers, enzyme inhibitors, enzyme substrate or cofactors, preservatives, stabilizers, detergents, sugars, facilitators, activators, oxidants, reductants, or any other additives desired for a particular purpose.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example, an absorbent pad in fluid communication with the first end, the second end, or both ends of the porous member of the present invention. The absorbent pad can be made of any material capable of holding an aqueous or liquid sample in fluid communication with the porous member such that the sample can flow evenly through the porous member by capillary action.
- In addition to the components of the present invention described above, other components may be included in the present invention, such as for example a housing. The housing may be made of any rigid material capable of housing the components of the present invention The housing may be preferably made of an impermeable material that contains the liquid sample and the components of the present invention inside the device without allowing for any leaks for permeation. The housing may be made of, for example, plastic, glass, metal, rubber, or any other non-permeable material. The housing may preferably include one or more apertures. The housing may include, for example, an aperture located at the first end of the porous member to act as an inlet for introduction of the test sample to the porous member. The housing may also include an aperture located on the area of the control zone, located near the second end of the porous member to allow for detection of whether or not the test has reached completion.
- This Example provides a device detection of methycillin resistant Staphylococcus aureaus (“MRSA”). Pastorex Staph Plus (Biorad, part #65356) was employed to demonstrate feasibility and utility of the present invention. As an experimental control, a slurry of MRSA was run on the slide agglutination assay per the manufacturer's instructions. Moderate agglutination was observed in the well containing the mixture of test latex and MRSA, and no agglutination was observed in the well containing a mixture of MRSA and the control latex.
- To identify a suitable chromatographic media, the suspension of red latex test particle was applied to several types of media including nitrocellulose and porous polyethylene. A porous polyethylene membrane (Porex, part #181071) with suitable chromatographic properties was selected. When untreated particles were applied to this membrane, the pore space volume was uniformly filled with particles producing a dark red color throughout the membrane. An 18 millimeter wide ribbon of this membrane was laminated to a clear lexan support with double sided tape and cut into 6 millimeter wide test strips.
- Next, 25 microliters of test particles and 25 microliters of control latex particles were dispensed into separate new tubes. To each was added 0.5 microliters of yellow food coloring to impart contrasting color to the clear solution 5 microliters of heat killed MRSA was added to each tube, mixed and allowed to incubate for 1 minute at room temperature. Twelve microliters of either test or control mixture was added to the proximal ends of separate membrane strips and the strips were rapidly filled by capillary action. As with the untreated latex above, the control latex was not excluded from the membrane and a dark red/orange color was clearly visible at the end of the strip distal to the area of sample application. However, the test latex, which had aggregated, was excluded from entering the membrane and only the yellow contrasting color of the solution was visible at the distal end of the strip.
- To test the relative sensitivity of this assay system a ten-fold dilution of the MRSA slurry is tested both on the chromatographic procedure described above and by the slide agglutination assay. The diluted sample produced the same distinct yellow color indicating separation by agglutination on the chromatographic media. However, only faint and hence conclusive aggregation was observed on the slide agglutination assay. This would suggest an initial improvement in sensitivity inherent to the invention even with a single chromatographic system.
- This Example provides a device detection of Streptococcus agalactiae (“Strep A”). Construction of the agglutination separation strip:
- Materials for Construction of the Agglutination Separation Strip:
-
- Millipore nitrocellulose: length 18 millimeter,
width 6 millimeter (Millipore, Inc., Part #PK002057; 100% nitrocellulose membrane) - Bridge pads (Ahlstrom 1281; 90% cellulose fiber, 10% rayon with traces of polyacrylamide wet strength resin and polyacrylamide dry strength resin)
- Absorbent pad (Ahlstrom 939; 100% cellulose with traces of polymide wet strength resin)
- Lexan backing (Lexan #8010)
Materials for Construction of the Antibody Capture Strip: - Millipore nitrocellulose: length 18 millimeter,
width 6 millimeter (Millipore, Inc., Part #PK002057) - Bridge pads (Ahlstrom 1281)
- Conjugate pads (Hollingsworth & Vose, H&V 7304 Nonwoven; polyester)
- Lexan backing (Lexan #8010)
Agglutination Procedure: - Remove gold conjugate pad from
Configuration 1 and add to a 12×75 millimeter glass tube - Add 100 microliter of Strep A moderate control to the pad and incubate for the desired time After incubation transfer available liquid to the lower bridge pad and allow chromatography to occur for desired time
- Read patient line or results at the interface where the bridge pad joins the nitrocellulose; if the analyte is present in the sample, agglutination with the gold particles will occur and deposit at the interface
Antibody Capture Procedure: - Add 100 microliter of Strep A moderate control to the bottom bridge pad of
configuration 2 - Allow lateral chromatography to proceed for desired time
- Read results at the antibody capture line
Results (Experiment 1): - For non-specific binding (“NSB”), use for moderate Strep A positive control buffer
Agglutination Antibody Capture Visual signal intensity: 2+ 2 NSB: — —
Results (Experiment 2): - Preincubation kinetic study
-
- Moderate Strep A positive control
- NSB as in
Experiment 1, above
- All chromatography at 15 minutes
Preincubation time(min) Agglutination Capture Antibody 5 1 .5 7 1 .5 10 1 .5 12 1 .5 15 1 .5 17 1 .5 20 1 .5 NSB at 20 minutes — — - Referring to
FIG. 1A the present invention includes anoptional absorbent 11 pad at one end in fluid communication with the firstporous member 12. The first porous member is in fluid communication with the secondporous member 14, forming ajunction 13. An important aspect of the firstporous member 12 of the present invention is its ability to substantially allow any detectable specific binding reagent to pass freely through the firstporous member 12 only when the detectable specific binding reagent is not bound to any analyte. An important aspect of the secondporous member 14 is its ability to substantially arrest or impede the flow of any bound detectable specific binding reagent from the secondporous member 14, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents. The present invention may optionally include anabsorbent pad 14 in fluid communication with the secondporous member 14. In one embodiment, the present invention may include a thirdporous member 16, which includes the detectable specific binding reagents and is used to contact the test device in order to run the test. - Referring to
FIG. 1B the present invention includes aporous member 16, which has the ability to substantially allow any detectable specific binding reagent to pass freely through theporous member 16 only when the detectable specific binding reagent is not bound to any analyte and substantially arrest or impede the flow of any bound detectable specific binding reagent from theporous member 16, especially when the bound detectable specific binding reagent form coagulates, agglutinates, clumps, or aggregates of bound detectable specific binding reagents. - This Example refers to
FIG. 2A andFIG. 2B , and provides a device detection of human chorionic gonadotropin (“hCG”). - 1 means absorbent pad
- 2 means nitrocellulose or other membrane
- 3 means bridge pad
- 4 means conjugate pad
- 5 means sample pad
- 4,5 means pad is used both for conjugate application and sample application, either together or separately.
- 6 means clear plastic backing material
Background: - The hCG (human chorionic gonadotropin) system is an excellent candidate for development of the agglutination process due to the characteristics of hormones in urine.
- Hormone levels are meaningful at very low concentrations, so detection of 10-60 mIUs needs to be effective.
- Procedure:
- First, the test strips were constructed as shown above, and stored in a dry container.
- Second, the test reagents were obtained, including:
-
- 2 vials containing hCG in urine at a concentration of 10 mIU and 60 mIU.
- A sample of 440 nm latex particles adsorption coated with Goat-anti-hCG antibodies, in a 1% PEG/PBS buffer.
- Third, test runs were performed by mixing the liquid latex-antibody solution mixed with the target antigen in a test tube, for zero to 10 minute incubations:
-
- The latex/antibody was added to a test tube containing different levels (mIU) of hCG, or a negative control.
- The mixture was added to one of the above agglutination devices above at the sample pad location.
- In various tests, at either zero time or at a later time the sample was “pushed” with an appropriate buffer, either PBS or Malaria buffer.
- The sample was then read, noting the time at which the reading was taken and the signal intensity noted, or lack of signal.
Results:
- 1.—5 ul of 440 nm latex+Goat-anti-hCG-beta antibody
-
- 40 ul of 60 mIU hCG
- Mix in test tube
- 10 minute incubation
- Using agglutination device “A” above, using SR nitrocellulose for membrane (2).
- Apply all 45 ul to sample application pad (4,5)
- Sample was ‘pushed’ with PBS.
- After 10 minutes the signal was 1+.
- 2.—5 ul of 440 nm latex+Goat-anti-hCG-beta antibody
-
- 40 ul of 60 mIU hCG
- Mix in test tube
- 1 minute incubation
- Using agglutination device “A” above, using SR nitrocellulose for membrane (2).
- Apply all 45 ul to sample application pad (4,5)
- Sample was ‘pushed’ with PBS.
- After 10 minutes the signal was 0.25+
- 3.—3 ul of 440 nm latex+Goat-anti-hCG-beta antibody
-
- 40 ul of PBS
- Mix in test tube
- 1 minute incubation
- Using agglutination device “A” above, using SR nitrocellulose for membrane (2).
- Apply all 45 ul to sample application pad (4,5)
- Sample was ‘pushed’ with PBS.
- After 10 minutes the signal was 0, no NSB.
- 4.—2 ul of 440 nm latex+Goat-anti-hCG-beta antibody
-
- 40 ul of (a) PBS, (b) 10 mIU hCG, (c) 60 mIU hCG
- Mix in test tubes
- 10 minute incubation
- Using agglutination device “A” above, using ST-type nitrocellulose for membrane (2).
- Apply all 42 ul to sample application pad (4,5)
- Sample was ‘pushed’ with Malaria buffer.
- After 5 minutes the signal was (a) 0, no NSB, (b) 1−, and (c) 1.
- 5.—5 ul of 440 nm latex+Goat-anti-hCG-beta antibody
-
- 40 ul of (a) negative urine, (b) 10 mIU hCG, (c) 60 mIU hCG
- Mix in test tube
- 2 minute incubation
- Using agglutination device “A” above, using SR nitrocellulose for membrane (2).
- Apply all 45 ul to sample application pad (4,5)
- Sample was ‘pushed’ with Malaria buffer.
- After 5 minutes the signal was (a) 0, (b) 1, (c) 1+.
- 6.—5 ul of 440 nm latex+Goat-anti-hCG-beta antibody
-
- 40 ul of (a) negative urine, (b) 10 mIU hCG, (c) 60 mIU hCG
- Mix in test tube
- 3 minute incubation
- Using agglutination device “A” above, using SR nitrocellulose for membrane (2).
- Apply all 45 ul to sample application pad (4,5)
- Sample was ‘pushed’ with Malaria buffer.
- After 5 minutes the signal was (a) 0, (b) 1/1+, (c) 1/1+.
- 7.—5 ul of 440 nm latex+Goat-anti-hCG-beta antibody
-
- 40 ul of (a) negative urine, (b) 60 mIU hCG
- Mix in test tube
- No incubation
- Using agglutination device “A” above, using SR nitrocellulose for membrane (2).
- Apply all 45 ul to sample application pad (4,5)
- Sample was ‘pushed’ with Malaria buffer.
- After 5 minutes the signal was (a) 0.5+, (b) 2+/3−.
- 8.—440 nm latex+Goat-anti-hCG-beta antibody mixed with an equal amount of drying buffer (2% Tween-20), then dried onto the sample/conjugate pad.
-
- 40 ul of (a) negative urine, (b) 60 mIU hCG
- Using agglutination device “B” above, using SR nitrocellulose for membrane (2).
- Apply the 40 ul to sample/conjugate pad (4,5)
- Note: there is no incubation time, this system mixes and runs instantly.
- Sample was ‘pushed’ with Malaria buffer.
- After 5 minutes the signal was (a) 0.5, (b) 1/1+.
- This Example refers to
FIG. 3A andFIG. 3B , and provides a device for dual antigen detection assay. - The card based assay is based on the standard ICT card assay design with an additional test strip configured in the opposite direction (
FIG. 3A ): - 1 means window for viewing results of antigen A
- 2 means window for viewing results of antigen B
- 3 means absorbent pad (Ahlstrom 939; 100% cellulose with traces of polymide wet strength resin)
- 4 means Millipore nitrocellulose: length 18 millimeter,
width 6 millimeter (Millipore, Inc., Part #PK002057) or Porex Corp. membrane research AB (fast flow) and research ACB (slow flow) with Porex proprietary chemical particle size exclusion barrier. - 5 means bridge pad (Ahlstrom 1281; 90
% cellulose fiber 10% rayon with traces of polyacrylamide wet strength resin and polyacrylamide dry strength resin) - 6 means conjugate pad (Hollingsworth & Vose, H&V 7304 Nonwoven; polyester)
- 7 means sample pad (Ahlstrom 1281; 90% cellulose fiber, 10% rayon with traces of polyacrylamide wet strength resin and polyacrylamide dry strength resin)
- 8 means sample insertion port (swab based)
- The cassette based assay design has the configuration of the test strips resembling that of (
FIG. 3B ): - 9 means window for viewing results of antigen A
- 10 means sample port
- 11 means window for viewing results of antigen B
Materials for Construction of Agglutination Dual Antigen Assay - Millipore nitrocellulose: length 18 millimeter,
width 6 millimeter (Millipore, Inc., Part #PK002057: 100% nitrocellulose membrane) or Porex Corp. membrane research AB (fast flow) and research ACB (slow flow) - Bridge pads (Ahlstrom 1281; 90% cellulose fiber, 100% rayon with traces of polyacrylamide wet strength resin and polyacrylamjde dry strength resin)
- Absorbent pad (Ahlstrom 939; 100% cellulose with traces of polyacrylamide wet strength. resin)
- Conjugate pads (Hollingsworth & Vose, H&V 7304 Nonwoven; polyester) Lexan backing (Lexan #8010)
- Sample Pad (Ahlstrom 1281; 90% cellulose fiber, 10% rayon with traces of polyacrylamide wet strength resin and polyacrylamide dry strength resin)
Procedure - For System of
FIG. 3A and System ofFIG. 3B the samples are added to the sample pad in 100 microliter volumes. - If any further flow of cojugate is required, 1×PBSA (pH 7.4) is added to the sample pad.
- Allow to run for 15 minutes
- Depending on the system, results can either be read at the interface point between the bridge pad and nitrocellulose, or at the 300 nanometer exclusion chemical barrier applied to the Porex membrane.
- The designations “first”, “second”, or “third” as used throughout this description of the present invention are merely for purposes of convenience and are used in order to avoid any confusion. These terms are not used for and do not imply any special ordering or arrangements in terms of priority, element structure, reactivity, or the like.
- All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. Various changes and departures may be made to the present invention without departing from the spirit and scope thereof. Accordingly, it is not intended that the invention be limited to that specifically described in the specification or as illustrated in the drawings, but only as set forth in the claims.
Claims (126)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/060,113 US20050221386A1 (en) | 2004-02-17 | 2005-02-17 | Chromatographic exclusion agglutination assay and methods of use thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US54541904P | 2004-02-17 | 2004-02-17 | |
US11/060,113 US20050221386A1 (en) | 2004-02-17 | 2005-02-17 | Chromatographic exclusion agglutination assay and methods of use thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US54541904P Continuation | 2004-02-17 | 2004-02-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
US20050221386A1 true US20050221386A1 (en) | 2005-10-06 |
Family
ID=34886150
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/060,113 Abandoned US20050221386A1 (en) | 2004-02-17 | 2005-02-17 | Chromatographic exclusion agglutination assay and methods of use thereof |
Country Status (5)
Country | Link |
---|---|
US (1) | US20050221386A1 (en) |
EP (1) | EP1718968A4 (en) |
JP (1) | JP2007523348A (en) |
CN (1) | CN101019023A (en) |
WO (1) | WO2005079420A2 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090289201A1 (en) * | 2008-05-20 | 2009-11-26 | Rapid Pathogen Screening, Inc. | Combined visual/fluorescence analyte detection test |
US20100297611A1 (en) * | 2008-05-20 | 2010-11-25 | Rapid Pathogen Screening, Inc. | Method and Device For Combined Detection Of Viral And Bacterial Infections |
US20110117672A1 (en) * | 2006-03-21 | 2011-05-19 | Magnisense Technology Limited | Magnetic immunochromatographic test method and device |
JP2013174612A (en) * | 2005-11-12 | 2013-09-05 | Platform Diagnostics Ltd | Agglutination assay |
US8815609B2 (en) | 2008-05-20 | 2014-08-26 | Rapid Pathogen Screening, Inc. | Multiplanar lateral flow assay with diverting zone |
US8962260B2 (en) | 2008-05-20 | 2015-02-24 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
US10379121B2 (en) | 2008-05-20 | 2019-08-13 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
US10808287B2 (en) | 2015-10-23 | 2020-10-20 | Rapid Pathogen Screening, Inc. | Methods and devices for accurate diagnosis of infections |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5789154A (en) * | 1993-10-12 | 1998-08-04 | Cornell Research Foundation, Inc. | Liposome-enhanced immunoassay and test device |
US5824268A (en) * | 1995-05-19 | 1998-10-20 | Universal Health Watch, Inc. | Rapid self-contained assay format |
US6472226B1 (en) * | 1998-10-02 | 2002-10-29 | Genosis Limited | Assay using porosity-reduction to inhibit migration |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5236826A (en) * | 1985-12-10 | 1993-08-17 | Murex Corporation | Immunoassay for the detection or quantitation of an analyte |
JPS63305251A (en) * | 1987-06-05 | 1988-12-13 | Dai Ichi Pure Chem Co Ltd | Immunoassay utilizing latex aggregation reaction |
EP0882224A4 (en) * | 1996-02-22 | 1999-06-09 | Dexall Biochemical Labs Inc | Non-captive substrate liquid phase immunoassay |
US6753189B1 (en) * | 1998-06-04 | 2004-06-22 | Mizuho Medy Co., Ltd. | Detection apparatus and method for the same |
-
2005
- 2005-02-17 US US11/060,113 patent/US20050221386A1/en not_active Abandoned
- 2005-02-17 CN CNA2005800105816A patent/CN101019023A/en active Pending
- 2005-02-17 WO PCT/US2005/004896 patent/WO2005079420A2/en active Application Filing
- 2005-02-17 JP JP2006554175A patent/JP2007523348A/en not_active Withdrawn
- 2005-02-17 EP EP05723144A patent/EP1718968A4/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5789154A (en) * | 1993-10-12 | 1998-08-04 | Cornell Research Foundation, Inc. | Liposome-enhanced immunoassay and test device |
US5824268A (en) * | 1995-05-19 | 1998-10-20 | Universal Health Watch, Inc. | Rapid self-contained assay format |
US6472226B1 (en) * | 1998-10-02 | 2002-10-29 | Genosis Limited | Assay using porosity-reduction to inhibit migration |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013174612A (en) * | 2005-11-12 | 2013-09-05 | Platform Diagnostics Ltd | Agglutination assay |
US9329181B2 (en) * | 2006-03-21 | 2016-05-03 | Magnisense Technology Limited | Magnetic immunochromatographic test method and device |
US20110117672A1 (en) * | 2006-03-21 | 2011-05-19 | Magnisense Technology Limited | Magnetic immunochromatographic test method and device |
US8962260B2 (en) | 2008-05-20 | 2015-02-24 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
US8470608B2 (en) | 2008-05-20 | 2013-06-25 | Rapid Pathogen Screening, Inc | Combined visual/fluorescence analyte detection test |
US8815609B2 (en) | 2008-05-20 | 2014-08-26 | Rapid Pathogen Screening, Inc. | Multiplanar lateral flow assay with diverting zone |
US20090289201A1 (en) * | 2008-05-20 | 2009-11-26 | Rapid Pathogen Screening, Inc. | Combined visual/fluorescence analyte detection test |
US20100297611A1 (en) * | 2008-05-20 | 2010-11-25 | Rapid Pathogen Screening, Inc. | Method and Device For Combined Detection Of Viral And Bacterial Infections |
US9372192B2 (en) | 2008-05-20 | 2016-06-21 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
US9910036B2 (en) | 2008-05-20 | 2018-03-06 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
US9933423B2 (en) | 2008-05-20 | 2018-04-03 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
US10379121B2 (en) | 2008-05-20 | 2019-08-13 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
US10408835B2 (en) | 2008-05-20 | 2019-09-10 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
US10808287B2 (en) | 2015-10-23 | 2020-10-20 | Rapid Pathogen Screening, Inc. | Methods and devices for accurate diagnosis of infections |
Also Published As
Publication number | Publication date |
---|---|
WO2005079420A2 (en) | 2005-09-01 |
EP1718968A2 (en) | 2006-11-08 |
JP2007523348A (en) | 2007-08-16 |
CN101019023A (en) | 2007-08-15 |
WO2005079420A3 (en) | 2007-01-18 |
EP1718968A4 (en) | 2008-07-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8389209B2 (en) | Test device for rapid diagnostics | |
KR102209489B1 (en) | Method and device for combined detection of viral and bacterial infections | |
JP4933258B2 (en) | Device for detecting multiple analytes in a sample | |
US20120107956A1 (en) | Indirect lateral flow sandwich assay | |
US20050221386A1 (en) | Chromatographic exclusion agglutination assay and methods of use thereof | |
JP2012524277A (en) | Extending test strip dynamic range | |
CN111077309B (en) | Immunoassay with capture conjugate | |
KR20060109595A (en) | Improved lateral flow immunoassay and device therefor | |
JP7451403B2 (en) | Lateral flow assays and methods for detecting high concentrations of analytes | |
CN111033237B (en) | Sandwich assay for measuring analytes, including high concentrations of analytes, using decreasing signal portions of dose-response curves | |
EP3870205B1 (en) | Lateral flow assays for differential isotype detection associated with zika virus | |
JP2009192222A (en) | Immunoassay method | |
JP2010032396A (en) | Biosensor | |
KR102526986B1 (en) | A rapid diagnostic kit using immunochromatography | |
US20070042504A1 (en) | Method for determining substance or substances in liquid sample | |
JP2023165878A (en) | Immuno-chromatography amplifying surface plasmon resonance by using carrier particle | |
US20130337580A1 (en) | Immunoassay Device and Method |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: BINAX, INC., MAINE Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:TURNER, NATHAN B.;PIASIO, ROGER N.;PIASIO, ERIK R.;REEL/FRAME:018264/0822;SIGNING DATES FROM 20060828 TO 20060829 |
|
AS | Assignment |
Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT,MAR Free format text: FIRST LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY;INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION;ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION;AND OTHERS;REEL/FRAME:019477/0268 Effective date: 20070626 Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT, MA Free format text: FIRST LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY;INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION;ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION;AND OTHERS;REEL/FRAME:019477/0268 Effective date: 20070626 |
|
AS | Assignment |
Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT,MAR Free format text: SECOND LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY;INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION;ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION;AND OTHERS;REEL/FRAME:019477/0808 Effective date: 20070626 Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT, MA Free format text: SECOND LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY;INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION;ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION;AND OTHERS;REEL/FRAME:019477/0808 Effective date: 20070626 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |